Episomal vector and uses thereof

ABSTRACT

The invention relates to a recombinant vector for stable persistence of erogenous DNA in a eukaryotic host cell, and the uses of the recombinant vector for long-term stable production of a gene product in the host cell, the vector including the minimal origin of replication of papillomavirus and the minichromosomal maintenance element of papillomavirus.

FIELD OF THE INVENTION

[0001] The invention relates in general to episomal vectors.

BACKGROUND OF THE INVENTION

[0002] In lower organisms, such as prokaryotes and budding yeast,replication origins contain welldefined cis-sequences called“replicators” and interaction of these sequences with a specificinitiator protein complex leads to the initiation of DNA synthesis inthese cells (Jacob et al., 1963; Stillman, 1994 and references therein;DePamphilis, 1993). Extrachromosomal replicators, generally, in additionto their origin function, encode functions that assure equaldistribution of replicated molecules (i.e., partitioning) betweendaughter cells at cell division. For prokaryotic plasmids thesepartitioning functions are well studied and can be provided by severaldifferent mechanisms in bacterial cells (Nordström, 1990). In higherorganisms, less is known about mechanisms for partitioning ofextrachromosomal replicators. For artificial plasmids in yeast,chromosomal centromeres can provide this function. In metazoan cells,one well studied example of a stable extrachromosomal replicatorexists—the latent origin oriP from Epstein-Barr Virus (EBV). Themaintenance function of EBV requires the viral replication factor EBNA-1and a series of binding sites for EBNA-1 termed the family of repeats(FR). A model that has been suggested for the function of the EBNA-1/FRcombination is that EBNA-1 bound to FR provides physical retention ofthe oriP plasmids in the cell nucleus (Krysan et al., 1989).

[0003] Papillomaviruses are also capable of stable extrachromosomalreplication. Infection and transformation of the cells bypapillomaviruses follows single hit kinetics. (Dvoretzky et al., 1980).Papillomavirus genomes are maintained as multicopy nuclear plasmids intransformed cells. The viral life-cycle can be viewed as three stages(Botchan et al., 1986). First, following initial entry, thepapillomaviral genome is amplified in the cell nucleus, i.e., viral DNAis synthesized faster than chromosomal DNA and the copy-number isincreased. The second stage represents maintenance of the viral DNA at aconstant copy-number and latent phase of the viral infection isestablished. During the third, vegetative, stage of the viral life-cycleviral DNA amplification is initiated again, late proteins aresynthesized and viral particles are assembled.

[0004] The E1 and E2 proteins are the only viral factors required forinitiation of papillomavirus DNA replication (Ustav and Stenlund 1991;Ustav et al., 1991; Yang et al., 1991; Chiang et al., 1992; Kuo et al.,1994). A similar, if not identical, set of cellular replication factorsand enzymes, in addition to viral initiator proteins, is utilized bySV40 (Tsurimoto et al., 1990; Weinberg et al., 1990) and BPV-1 ( Mülleret al., 1994) at the origin of replication to initiate DNA synthesis.Analysis of the essential cis-sequences shows that the BPV-1 minimalorigin (Ustav et al., 1993) resembles a typical eukaryotic origin ofreplication (DePamphilis, 1993) and it has been suggested that thissimilarity could also be extended to the mechanisms of replication ofall papovaviruses (Nallaseth and DePamphilis, 1994; Bonne-Andrea et al.,1995). However, the ability of the papillomaviruses to persist asplasmids distinguishes papillomaviruses from other papovaviruses. It hasbeen known for more than 10 years that BPV-1 replicates in transformedcells as a multicopy nuclear plasmid, which can persist in the tissueculture cells over long periods of time (Law et al., 198 1). Thisindicates that papillomaviruses have efficient mechanisms forsegregation, i.e., control of copy-number and partitioning, in thetransformed cells.

[0005] The role of viral factors, cis-acting sequences and cellularfactors in long-term persistence of papillomaviruses, which relates tothe segregation functions of viral DNA, is not clearly understood. Thatis, the regions of the viral genome which specify copy number are notidentified in the prior art; nor are the regions of the viral genomewhich participate with the host cell to ensure proper segregation of theviral genome during partitioning. Much more is understood with respectto the initial amplification stage of the papillomavirus life-cycle.

[0006] Bovine Papillomavirus (BPV) and Human Papillomaviruses (HPVs)persist as stably maintained plasmids in mammalian cells. Transientassays, i.e., on the order of several hours to 3-4 days, have been usedto define the minimal origin of replication (MO) which is required fortransient replication in BPV (Ustav et al., EMBO J, 10, 4231-4329,199 1) and for several HPV subtypes. Two trans-acting factors encoded byBPV and HPVs, namely E1 and E2, have been identified in transient assayswhich are necessary to mediate replication in many cell types via MO(Ustav et al., EMBO J., 10, 449-457 (1991); Ustav et al., EMBO J, 10,4231-4329, (1991); Ustav et al., PNAS, 90, 898-902 (1993).) E1 and E2from BPV will replicate via the BPV MO and via the MO of many HPVsubtypes. (Chiang et al., PNAS, 89, 5799-5803 (1992). E1 and E2 from HPVwill replicate via the BPV MO and via the MO of many HPV subtypes.(Chiang et al., PNAS, 89, 5799-5803 (1992). Replication of plasmidscontaining the above elements is high level but transient in eukaryoticcells. Plasmid loss is rapid in the presence and absence of selectivepressure.

[0007] The papillomavirus life cycle has been the subject of muchresearch. Different portions of the viral genome have been tested inshort-term, i.e., transient, transcription or replication assays. See,for example, Szymanski el al., 1991, Jour. Virol. 11:5710; Vande Pol etal., 1990., Jour. Virol 64:5420; Sowden et al., 1989, Nucl. Acids Res.17:2959; Stenlund, 1987, Science 236:1666; Sedman et al., 1995, Eur.Jour. Mol. Biol. 14:6218; Haugen et al., 1988, Eur. Jour. Mol. Biol.7:4245; and Kuo et al., 1994, Jour. Biol. Chem. 269:24058.

[0008] The BPV 69% transforming region has been used to introduce therat preproinsulin gene into mouse cells (Sarver et al., 1981, Mol. Cell.Biol. 6:486).

[0009] The PMS1 and PMS2 regions of BPV have been reported to“independently support” extrachromosomal replication of the Tn5 neomycingene in cells that provide viral factors in trans. PMS-1 (plasmidmaintenance sequence-1) is localized within a 521 bp region mapping atpositions 6945-7476 of the BPV genome, and PMS-2 has been localized to a140 bp region within the putative open reading frame for the E1 protein,which maps at positions 1515-1655 of the BPV genome. It has beenreported that recombinant plasmids carrying either of the PMS elementsare unrearranged and stably maintained at a constant copy number. Inaddition, E1, E6 and E7 are identified as candidate factors for transregulation of the plasmid state. See Lusky et al., 1984, Cell 36:391,and Lusky et al., 1986, Jour. Virol. 11:729.

[0010] Woo et al., W094/12629 report a vector containing a papillomavirus origin of replication, the “vector maintenance sequence” describedin Lusky et al., 1984, supra, a therapeutic nucleic acid, and an E2 genesequence or an E1 /E2 chimeric gene. Woo et al. suggest that such avector may be tested for stable episomal maintenance over a period of2-30 days post-transfection. The “vector maintenance sequence” of Luskyet al., 1984, which is described in Woo et al., is shown herein not tobe capable of providing long-term vector persistence.

[0011] Mutations in the E2 gene have a pleiotropic effect on viral genefunctions, including oncogenic transformation. These effects may be theresult of the requirement for E2 expression to regulate viraltranscription (see DiMaio and Neary, 1989, The Genetics of bovinepapillomavirus type 1 papillomaviruses and human cancer. (Ed. N.Pfister), CRC Press, Boca Raton, Fla.). The BPV-1 E2 protein has beenshown to activate viral enhancers in trans (Spalholz et al., 1985, Cell42:183). The E2 open reading frame has been shown to encode asite-specific DNA binding protein that can bind to several sites withinthe E2 responsive enhancers 1 and 2 (Androphy et al., 1987, Nature325:70; Moskaluk et al., 1987, Proc. Nat. Aca. Sci. 84:1215). E2recognition sites that have been studied to date include the sequencemotif ACCN6GGT, where N is any nucleotide (Hawley-Nelson et al., 1988,Eur. Jour. Mol. Biol. 7:525; Hirochika et al., 1988, Genes Dev. 2:54;McBride, 1988, Eur. Jour. Mol. Biol. 7:553; Moskaluk et al., 1988a, Prc.Nat. Aca. Sci. 85:1826), and it is suggested that E2 binds thispalindrome as a dimer (Dostani et al., 1988, Eur. Mol. Biol. Org. Jour.7:3 807; McBride et al., 1989, Proc. Nat. Aca. Sci. 86:510). Not all ofthese sites appear to bind E2 with the same strength. Sites having themotif ACCGN4CGGT appear to bind better than sites that deviate in thefourth and ninth bases (Hawley-Nelson et al., 1988, supra; Moskaluk etal., 1988b, supra). It also appears that some of the target sites forthe protein have different capabilities for activation in vivo (Harrisonet al., 1987, Nucl. Acids. Res. 15:10267; Haugen et al., 1987, Eur.Jour. Mol. Biol. 6:145; Spalholz et al., 1987, Jour. Virol. 61:2128). Liet al. (1989, Genes & Develop. 510) analyze 17 E2 binding sites in theBPV-I genome and show that affinities for E2 vary over a 300-fold range.Li et al. also find that the presence of the conserved consensusACCGN4CGGT did not necessarily guarantee that the binding site would bestronger than one with a deviant base, and suggest that the presence ofthis palindrome is not a sufficient parameter for predicting thestrength of a binding site.

[0012] A truncated form of E2 protein exists which is defective intranscriptional activation and competent in viral replication.

[0013] Dowhanick et al., 1995, Jour. Virol. 69:7791, describe an E2deletion mutant containing residues 1-218 of the protein which is saidto retain a DNA replication function, but is defective intranscriptional trans-activation. Also described are several E2 pointmutants (331 and 344) which are defective in both transcriptionaltransactivation and DNA binding.

[0014] In addition, subsequent to Applicant's disclosure of E2 pointmutants which are defective in transcriptional activation andreplication competent in the subject priority document, which E2 mutantsare also described in Abroi et al., 1996, Jour. Virol. 70:6169,additional similar, if not identical in some instances, E2 point mutantshave been identified. Ferguson and Botchan, 1996, Jour. Virol. 70:4193,describe mutations at amino acids 73 and 74 which retained replicationfunction but failed to activate transcription”. Sakai et al., 1996,Jour. Virol. 70:1602, describe three point mutants (R37A, 173A, andW92A) in HPV defective for transcriptional activation but retaining wildtype DNA replication activity in transient assays.

[0015] One object of the invention is to provide a recombinant vectorwhich, by virtue of the sequences it contains, is stably maintained andthus persists long-term in mammalian cells.

[0016] Another object of the invention is to provide a recombinantepisomal vector which is stabilized via regulatory sequences which arecontained within a relatively small amount of DNA.

[0017] Another object of the invention is to provide a cis-actingelement which confers long-term stability to a transiently replicatingeukaryotic episomal plasmid.

[0018] Yet another object of the invention is to provide an episomalgenetic element which replicates independently of the host cellchromosomal DNA, and is therefore not dependent upon regulatory controlof replication by the host cell genome.

[0019] Another object of the invention is to provide stable and reliableplasmid copy number in both G1 and G2 stages of the cell cycle.

[0020] Yet another object of the invention is to provide a recombinantvector which is stably maintained at a constant copy number for multiplecell generations.

[0021] Another object of the invention is to provide a recombinantvector which is able to persist over a long time period in eukaryotic,particularly mammalian cells, from which can be expressed a therapeutic,prophylactic, or marker gene.

[0022] Another object of the invention is to provide a recombinantvector which is not restricted as to its ability to be maintained in agiven cell type, but which is stably maintained in any one of numerousdiverse mammalian cell types.

[0023] Another object of the invention is to provide a recombinantvector containing sequences of viral origin which do not conferoncogenic properties to the transfected host cell, and is therefore safeto use in humans.

SUMMARY OF THE INVENTION

[0024] The invention is based on the discovery of a vector system whichpermits long-term persistence in episomal form in a mammalian cell, andin particular to the discovery of a minichromosomal maintenance element,which element confers stable persistence of extrachromosomal (i.e.,episomal) DNA in mammalian host cells.

[0025] The invention encompasses a method of obtaining long-term stableproduction of a gene product of interest in a host cell, comprisingproviding a host cell containing a vector comprising (A) a minimalorigin of replication of a papilloma virus, (B) a minichromosomalmaintenance element of a papilloma virus, and (C) a gene encoding thegene product, wherein the vector, when present in a mammalian host cell,persists in the cell for at least about 50 cell generations in dividingcells or for at least about 8 weeks in non-dividing cells undernonselective conditions without an appreciable loss of copy number.

[0026] The invention also encompasses a method of obtaining long-termstable production of a gene product of interest in a host cell,comprising providing a host cell containing a vector comprisingpapillomavirus sequences consisting essentially of (A) a papillomavirusE2 gene, (B) a minimal origin of replication of a papilloma virus,(C) aminichromosomal maintenance element of a papilloma virus, and (D) a geneencoding the gene product, wherein the vector persists in the cell forat least about 50 cell generations in dividing cells or for at leastabout 8 weeks in non-dividing cells under nonselective conditionswithout an appreciable loss of copy number.

[0027] The invention also encompasses a method of obtaining long-termstable production of a gene product of interest in a host cell,comprising providing a host cell containing a pair of vectors comprising(I) a first vector comprising papillomavirus sequences consistingessentially of (A) a papillomavirus E2 gene, (B) a minimal origin ofreplication of a papilloma virus, and (C) a minichromosomal maintenanceelement of a papilloma virus, and (II) a second vector comprisingpapillomavirus sequences consisting essentially of (A) a gene encodingthe gene product, (B) a minimal origin of replication of a papillomavirus, and (C) a minichromosomal maintenance element of a papillomavirus, wherein the vector persists in the cell for at least about 50cell generations in dividing cells or for at least about 8 weeks innon-dividing cells under nonselective conditions without an appreciableloss of copy number.

[0028] The invention also encompasses use of a recombinant vector forobtaining long term stable maintenance of erogenous DNA in a eukaryotichost cell wherein the recombinant vector comprises: a minimal origin ofreplication of a papillomavirus; a minchromosomal maintenance element ofa papillomavirus; and a heterologous DNA sequence encoding anexpressible gene.

[0029] Preferably, the time period over which the vector persists in thehost cell under nonselective conditions without an appreciable loss ofcopy number is 6 weeks, and most preferably 8 weeks or 12 weeks orlonger, or in terms of cell generations, 100 or 120 cell generations orlonger.

[0030] According to the claimed methods, long-term persistent vectorswill include one in which the minichromosomal maintenance elementconsists essentially of the region of BPV mapping to positions 7590 to7673; or wherein the minichromosomal maintenance element comprises (BPVE2 binding sites 6, 7 and 8) x, wherein x is 3 to 6 or wherein theminichromosomal maintenance element comprises at least 2 of the 3 E2binding sites 6, 7 and 8.

[0031] The invention therefore also encompasses a recombinant vector forstable long-term persistence of erogenous DNA in a mammalian host cell,the vector comprising a minimal origin (MO) of replication of apapillomavirus, a minichromosomal maintenance element (MME) of apapillomavirus, and a gene encoding a gene product of interest, whereinthe vector is defined hereinbelow (1-4).

[0032] 1. The vector comprising papilloma virus sequences consistingessentially of (A) a minimal origin of replication of a papilloma virus,(B) a minichromosomal maintenance element of a papilloma virusconsisting essentially of at least two of the three E2 binding sites 6,7, and 8, wherein the region of the vector comprising the minimal originof replication and minichromosomal maintenance element consists of a DNAsequence different from the natural papilloma virus sequence, andwherein the vector, when present in a mammalian host cell whichexpresses E1 and E2, persists in the cell for at least about 50 cellgenerations in dividing cells or for at least about 8 weeks innon-dividing cells under nonselective conditions without an appreciableloss of copy number.

[0033] 2. The vector comprising papilloma virus sequences consistingessentially of (A) a minimal origin of replication of a papilloma virus,and (B) a minichromosomal maintenance element of a papilloma virusconsisting essentially of multiple E2 binding sites, wherein thedistance between the minimal origin of replication and theminichromosomal maintenance element is less than about 1.0 kb, whereinthe vector, when present in a mammalian host cell which expresses E1 andE2, persists in the cell for at least about 50 cell generations individing cells or for at least about 8 weeks in non-dividing cells undernonselective conditions without an appreciable loss of copy number.

[0034] 3. The vector comprising papilloma virus sequences consistingessentially of (A) a minimal origin of replication of a papilloma virus,(B) a minichromosomal maintenance element of a papilloma virusconsisting essentially of the region of BPV mapping to about positions7590-7673 wherein the vector, when present in a mammalian host cellwhich expresses E1 and E2, persists in the cell for at least about 50cell generations in dividing cells or for at least about 8 weeks innondividing cells under nonselective conditions without an appreciableloss of copy number.

[0035] 4. The vector comprising papilloma virus sequences consistingessentially of (A) a minimal origin of replication of a papilloma virus,and (B) a minichromosomal maintenance element of a papilloma virusconsisting essentially of (BPV E2 binding sites 6, 7, and 8)x wherein xis 3-6, wherein the vector, when present in a mammalian host cell whichexpresses E1 and E2, persists in the cell for at least about 50 cellgenerations in dividing cells or for at least about 8 weeks innondividing cells under nonselective conditions without an appreciableloss of copy number.

[0036] As used herein, the term “consisting essentially of means that,with respect to papillomavirus sequences, those sequences which are bothnecessary and sufficient for long-term vector persistence without anappreciable loss of copy number.

[0037] In a preferred embodiment of the invention, a vector of theinvention will comprise papillomavirus sequences as well as othersequences relating to expression of a gene of interest. Thepapillomavirus sequences in the vector will preferably consistessentially of a papillomavirus MME and MO, and thus will not containpapillomavirus sequences that are not required for long-term stablepersistence in a eukaryotic host cell. The vectors thus advantageouslydo not contain papillomavirus sequences which are not both necessary andsufficient for long-term persistence in the episomal state. In addition,the vectors do not contain oncogenic sequences which are present in thepapillomavirus genome.

[0038] In preferred embodiments, the minichromosomal maintenance elementof a papillomavirus is from BPV; the minimal origin of replication ofpapillomavirus is from BPV; the papillomavirus E1 protein is from BPV;the papillomavirus E2 protein is from BPV.

[0039] Preferably, the vector further comprises a gene or genes encodingpapillomavirus E2 and/or E1 proteins, and the E2 gene most preferablyencodes a mutant form of E2 which is a point mutant that is replicationcompetent but defect in transcriptional activation. As used herein, a“point mutant” may refer to either a single amino acid change, orseveral individual amino acid changes (2, 3, 4 etc.) which togetherconfer the desired phenotype. A point mutant may be an amino acidsubstitution or a deletion or insertion.

[0040] One particularly useful form of a vector of the invention is arecombinant vector or vector system for stable persistence of erogenousDNA in a host cell, the vector comprising a minimal origin ofreplication of a papillomavirus, a minichromosomal maintenance elementof a papillomavirus, and one or both of the papillomavirus E1 and E2genes.

[0041] The invention also encompasses a mutant form of a papillomavirusE2 protein wherein the replication function of the protein is competentand the transcriptional activation function of the protein is defective,wherein the mutant form of E2 protein differs from the wild-type E2 in anucleotide point mutation which translates into an amino acidsubstitution.

[0042] Preferred E2 point mutants are mutated in an alpha helicaldomain, for example in alpha helix 2 or 3, as defined herein.

[0043] Additional preferred E2 point mutants useful according to theinvention are R37A, E74A, and D 122A and D143A/R172C.

[0044] A particularly striking feature of the invention is that thestable vectors of the invention are not restricted to the host cellspecificity of papillomavirus. This release from the naturalpapillomavirus host cell type restriction has been achieved by removingkey genetic elements of the papillomavirus genome from their nativecontext; for example, expression of the papillomavirus genes encoding E1and E2 proteins is not controlled by the promoters that are native tothese genes, but rather the E1 and E2 genes are placed under the controlof non-native, i.e., heterologous promoters, which are either functionalin a broad range of mammalian cells or tissues or are cell- ortissue-specific.

[0045] It is preferred according to the invention that the expressiblepapillomavirus gene encoding E1 or E2 include a structural gene encodingE1 or E2 operatively associated with regulatory sequences for expressionof the structural gene in a host cell. Such regulatory sequences willinclude a promoter and/or may optionally include an enhancer. Thepromoter is preferably a promoter that is non-native (i.e.,heterologous) to the E1 or E2 structural gene. The promoter is may befunctional in more than a single tissue type, i.e., one that is able toinitiate transcription in a broad range of tissue types, and thereforeunrestricted with respect to its tissue specificity. Alternatively, thepromoter may be functionally restricted to a single tissue type, andtherefore tissue-specific.

[0046] As used herein, tissue-specific and cell-type-restricted bothrefer to wherein a promoter is operable substantially in the sametissue-type or cell-type.

[0047] Preferred promoters comprise one of the thymidine kinase promoterand a strong promoter such as the SRalpha promoter. It is expected thata vector of the invention which includes tissue-specific regulatoryelements operatively associated with the E1 and/or E2 genes will becapable of long-term persistence only in those cell types in which theregulatory elements are functional.

[0048] In its most useful form, a recombinant vector of the inventionwill include an expressible gene of interest.

[0049] A vector of the invention which contains an expressible gene ofinterest contains not only a structural gene encoding a protein or RNAof interest, but also is operatively associated with regulatorysequences for expression of the structural gene in a host cell. Suchregulatory sequences may include not only a promoter, but alsoadditional regulatory sequences such as an enhancer, splice sites, andpoly-adenylation sequences. These regulatory elements that controlexpression of the structural gene promoter may be regulatory elementsthat are native to the structural gene (i.e., the control sequences thatare naturally associated with these genes in their native environment),or they may be non-native to the structural gene, and thereforeheterologous regulatory elements. These elements, particularly thepromoter, may be functional in more than a single tissue type or may befunctionally restricted to a single tissue.

[0050] It is expected that a vector of the invention which includes atissue-specific regulatory element that is operatively associated with astructural gene of interest will express that gene of interest only inthose host cell types in which the regulatory elements are functional(i.e., specific).

[0051] It is preferred according to the invention that the host celltype-restricted expression (i.e., tissue-specificity) of the structuralgene of interest be coordinated with the tissue-specificity of theregulatory elements operatively associated with the E1 and E2 genes.That is, one may envision that the tissue-specificity of E1, E2, andstructural gene of interest is the same. Alternatively, thetissue-specificity of E1 and E2 gene expression may be broader than thetissue-specificity of expression of the gene of interest, resulting in abroad host cell type range for long-term persistence of a vector of theinvention, and a more limited host cell type range for expression of thegene of interest. Alternatively, the tissue-specificity of E1 and E2gene expression may be quite limited (for example, to a single celltype), and the tissue-specificity of expression of the gene of interestbroad or unlimited, resulting in a limited host cell type in which avector of the invention can persist long-term, which in turn is thelimiting factor in determining the type of host cell in which the geneof interest is expressed.

[0052] In another preferred embodiment of a vector of the invention, thevector also includes a bacterial host cell origin of replication and agene encoding a selectable marker for preparation of vector DNA in abacterial host cell.

[0053] The invention also features host cells containing the vectorsherein described, such host cells being most preferably beingeukaryotic, and of mammalian origin, such as of muscle, gut, or brainorigin.

[0054] The invention also features a method of manufacture of a vector,which method includes culturing a host cell containing a vectordescribed herein. It is particularly preferred that such manufactureoccur in a lower eukaryotic cells, e.g., yeast or insect, or prokaryoticcells, e.g., bacterial cells such as E.coli or Salmonella. Therefor, thevector will further include an origin of replication of yeast, insect orbacterial origin, and one or more genes encoding a selectable marker,e.g., a gene encoding kanamycin resistance, for selection of cellscontaining the vector.

[0055] The invention also features a method of obtaining stableexpression of a gene of interest in a cell, comprising providing a hostcell as described above. The invention also features methods of treatinga disease stemming from a genetic defect, comprising administering atherapeutically effective or a prophylactic amount the vector of theinvention to a patient afflicted with the disease.

[0056] The invention also includes use of a recombinant vector of theinvention in the treatment of a disease.

[0057] The invention also encompasses a gene delivery system comprisinga vector of the invention in combination with a gene delivery vehicle,which may be of viral or non-viral origin.

[0058] The invention also encompasses a method of producing a protein orRNA of interest in a host cell or a transgenic animal, comprisingculturing a host cell under conditions which permit production of theprotein of interest, or providing a transgenic animal which produces theprotein, as described herein.

[0059] The invention also encompasses a mammalian model of disease, forscreening of drugs to treat the disease or for testing of therapeutic orprophylactic regimes, the mammalian model comprising a transgenic animalwhose cells contain a vector of the invention.

[0060] The invention also encompasses a transgenic animal containing anepisomal vector as described herein, the vector encoding a protein ofinterest.

[0061] As used herein, a “transgenic animal” refers to an animal,preferably a mammal, which contains in some, but not necessarily all, ofits cells an episomal vector, as described herein.

[0062] The invention also features kits for providing stable persistenceof a vector in a host cell, the kit comprising a vector or a host cellas described herein and packaging materials therefore.

[0063] A kit of the invention may also include a mutant E2 protein asdescribed herein or a gene encoding this protein, wherein the E2 mutantis thus provided for stable persistence of a vector in a host cell.

[0064] Uses and Advantages of the Invention are as Follows.

[0065] The invention is useful in in vivo and ex vivo human gene therapywhere correction of inherited or acquired genetic defects is desired.The invention also is useful in vaccination protocols where resistanceor immunity to infectious pathogens, such as HIV, Hepatitis C Virus,Hepatitis B virus, Herpes virus, parasitic pathogens such asTuberculosis and Leishmaniasis, and protozoans such as ameobicdysentery, is desired, or the elimination or induced quiescence ofaberrant cells, such as cancer cells, is considered beneficial.

[0066] Recombinant vectors of the invention are useful in that theypermit persistent expression of a therapeutic gene in both dividing andnon-dividing cells; for example, in differentiated cells, such as thosein brain, and muscle.

[0067] Recombinant vectors of the invention are also useful for highlevel transient expression in cells where desired, such as for cancertherapy or in vivo vaccination.

[0068] Both in vivo and ex vivo gene therapy strategies are possiblewith this vector system, including stable, multicopy gene maintenanceand expression, in haemopoietic and other stem cells, and in thecommitted and differentiated progeny of these cell types.

[0069] For human gene therapy, uses of the recombinant vectors of theinvention are not limited in terms of delivery of the vector to a cell.That is, vectors of the invention may be delivered to a cell vianon-viral or viral delivery systems. Delivery systems of non-viralorigin include those which employ cationic liposomes, where vector sizeconstraints do not limit the nature and number of plasmid vectorcomponents. Delivery systems of viral origin include viralparticle-producing packaging cell lines as transfection recipients forthe above E1/E2/M0/MME-containing plasmids into which viral packagingsignals have been engineered, such as those of adenovirus, herpesviruses and papovaviruses.

[0070] Recombinant vectors of the invention also are useful intransgenesis, including production of transgenic animals via pronuclearinjection, or embryonic stem cell transfection and embryo chimerageneration.

[0071] Recombinant vectors of the invention also are usefuil forpreparation of cell factories for stable, high level expression ofproteins of therapeutic value in cultured mammalian cells.

[0072] Further features and advantages of the invention will become morefully apparent in the following description of the embodiments anddrawings thereof and from the appended claims.

DESCRIPTION OF THE DRAWINGS

[0073] Before describing the invention in detail, the drawings will bebriefly described.

[0074]FIG. 1A is a scheme of the experimental protocol.

[0075]FIG. 1B is a short term replication assay for the plasmids in theCHO4.15 cells. Low molecular weight DNA was extracted from the CHO4.15cells transfected with the plasmids containing minimal origin or 2.5 kbBg1II fragment and analyzed by Southern blotting after digestion withDpnI and linearizing enzyme XbaI (lanes 1, 2) and HindIIl (lanes 3, 4).Lanes 1, 2—episomally replicating minimal origin containing plasmid DNAextracted 36 and 60 hours after transfection and lanes 3, 4—episomal DNAextracted 36 and 60 hours after transfection with the plasmid containing2.5 kb origin fragment.

[0076]FIG. 2A stable replication of the BPV-1 origin containing plasmidsin the CHO4.15 cell line. Representation of the BPV-1 fragment (BPVnucleotides 6945-1515) used in this experiment. The respective mutantsin this fragment are depicted and are further described in Materials andMethods. The following genetic elements are indicated: NCOR-1 and2—Negative Control Of Replication 1 and 2; PMS-1—Plasmid MaintenanceSequence; ORI—minimal origin of replication; E2RE1 andE2RE2—E2responsive enhancer 1 and 2; CE—constitutive enhancer; E1, E6,E7 and E8—respective ORF-s; P₇₁₇₅, P_(L), P₈₉, P₈₉₀—respectivepromoters; boxes indicate location of 14 E2 binding sites in thisfragment.

[0077]FIG. 2B is a southern blot analysis of stable cell lines for thepresence of episomal plasmids. The marker lanes 1, 2, 3—contain 100pages of linearized vector pNeo5′, pNeoBg140 and pNeoBg1II,respectively. Low molecular weight DNA was extracted from CHO4.15 cellsafter transfection and G418 selection (see Materials and Methods fordetails). The plasmids used were the vector plasmid pNeo5′ (lane 5),pNeoXhoI→HpaI with disrupted E1 protein binding site (lane 6), pNeoBg140(lane 7), pNeoΔNCORI with deleted Negative Control Region (lane 8),mutants Sma⁻, 775 and 576 with disrupted 5′-part of E1, E6 and E6/E7ORFs respectively (lanes 9-11), the wild type pNeoBg1II (lane 12), andthe minimal origin containing plasmnid pNeoMO (lane 13).

[0078]FIG. 3A shows stable extrachromosomal replication of the plasmidswith deletions in the URR in CHO4.15 cells. Representation of BPV—Ifragments with respective deletions.P₇₁₇₅, P_(L), P₇₉₄₀—respectivepromoters in this fragment, E2REI and E2RE2—E2 responsive enhancer, CEconstitutive enhancer, boxes indicate E2 protein binding sites. Endpoints of the respective deletions are given in Materials and Methods.Circle indicates the location of the minimal origin. Ability ofrespective deletion mutants to function in long term replication assayis indicated by (+) or (−).

[0079]FIG. 3B. Low molecular weight DNA was extracted from the cells,transfected with respective plasmids and selected for G418, digestedwith the linearizing enzyme HindIII and analyzed by Southern blot (lanes1-22). Lanes 23, 24 and 25 contain 100 pg of linearized pNeo5′ vector,pNeoBg140 and PNeoBg1II marker DNA.

[0080]FIG. 4A represents an analysis of state and copy-number of BPV-1origin containing plasmids in CHO4.15 cells. Copy-number measurement ofBPV plasmids stably replicating in CHO4.15 cells. Total DNA wasextracted from the stable cell lines and subjected for linearizationwith HindIII. Lanes 1-3 represent analysis of 2 μg of total DNA fromthree independent cell-lines, a series of plasmid dilutions forcopy-number reconstruction is included in lanes 4-6.

[0081]FIG. 4B shows plasmnid DNA, wherein a total of DNA 2 μg, cut withplasmid noncutter Apal, from four independent cell-lines is analyzed(lanes 1-4). Respective marker of uncut plasmid DNA is shown in lanes 5and 6.

[0082]FIG. 5 shows restoration of stable replication of the plasmids byan M consisting of oligomerized E2 binding sites. Low molecular weightDNA was extracted from the G418 resistant cells and linearized withHindIII (lanes 1-4) or with XbaI (lane 5). Lane 1 represents analysis ofthe DNA from the cells transfected with original D234/221 mutant withdeleted MME. Insertion mutants with E2REI (BPV nucleotides 7611-7673)cloned into D234/221 adding back 18 and 9 E2 binding sites restoredstable replication of the plasmid (lanes 2, 3). Mutant with oligomerized10 E2 binding sites cloned into D234/221 (lane 4) and mutant withdeleted PMS1 with inserted 10 oligomerized E2 binding sites (lane 5)replicate in the long term assay. Lanes 6-8 contain 100 ng of linearizedrespective marker DNA.

[0083]FIG. 6 shows the transformation efficiency (colonies per 100.000cells) for G418 resistance of the plasmids pNeoBg140 (Bg140),pNeoXhoI→HpaI (HpaXho), pNeoMO (MO) and vector pNeo5′. 500 ng of therespective plasmid DNA was electroporated into the cells and 84 hourslater cells were trypsinized, counted and plated. Complete mediumcontaining 450 μg/ml of G418 was used for selection. Colonies werecounted at the 10th day of selection. Presented values are average ofthree independent measurements. A. CHO4.15 cells, B. CHO212 cells and C.CHO49 cells.

[0084]FIG. 7A is a schematic illustration of a plasmid in which E1expression is under the control of the SR promoter.

[0085]FIG. 7B is a schematic illustration of a plasmid in which E1expression is under the control of the Thymidine kinase promoter.

[0086]FIG. 7C is a schematic representation of the plasmid maps shown inFIGS. 7A and 7B.

[0087]FIG. 8 represents a comparison of the stable replication modes ofBPV origin containing plasmid and chromosomal DNA in the CHO4.15 cells.BrdU labeling of the CHO4.15 cells carrying stably replicating pNeoBg140was done for a) and e)—3.5, b) and f)—9.5, c) and g)—15.0 and d) andh)—24 hours, respectively. Episomal-a), b), c), d) and total chromosomalDNA-e), f), g), h) were prepared at respective time points and analyzedas described in Materials and Methods. CsCl gradients were aliqoted,denatured, renatured and loaded onto the nylon filters by slot-blotterand hybridized with radioactive BPV-1 origin probe for episomal DNA andwith radioactive total CHO DNA for genomic DNA gradients. Intensity ofhybridization was quantitated by use of the Phosphoimager.

[0088]FIG. 9 shows the copy number of BPV-1 origin-bearing plasmids issimilar in G1 and G2 phases of the cell cycle. Southern blot analysis ofplasmid copy number in G1 and G2 phases of the cell cycle. Derivativesof CHO4.15 cell line with stably, extra chromosomally replicating BPV-1origin-containing plasmids were arrested in G1/S with mimosine orhydroxyurea and in G2 with hydroxyurea, followed by Hoecsht 33342treatment. Arrest was verified with FACS analysis, total DNA wasextracted and an equal amount of DNA was loaded onto each lane. Analysisof four different established cell lines is shown on the figure with 3parallels for each cell cycle arrest.

[0089]FIG. 10 shows transient replication of pSR alpha and pTk vectors(FIG. 7) in CHO (lane 1-4) and C333A (lanes 7-10) cell lines. 2micrograms of respective plasmnid was transfected into the cells byelectroporation, time points were taken 36 and 48 hourspost-transfection and low-molecular weight DNA was extracted, digestedwith DpnI and HindIII and analyzed by Southern blotting. Lanes 1-2 and7-8 represent transient replication of SR alpha vector in CHO and C33Acells respectively. Over-replication is clearly visible in case of bothcell lines. Lanes 3-4 and 9-10 represent transient replication of TKvectors in CHO and C33A cells respectively. Some over-replication isdetectable in C33A cells, whereas replication of TK vector in CHO cellsis weak, but without smear. The latter is characteristic for onion-skintype over-replication. Lanes 5-6 and 11 represent respectivemolecular-weight markers.

[0090]FIG. 11 shows beta-galactosidase activity in CHO cells transfectedwith equimolar amounts of vector plasmids. In plasmids pON260beta-galactosidase gene is expressed from immediate early CMV promoter.In other vector constructs beta-galactosidase is expressed from RSV LTR.

[0091]FIG. 12 shows beta-galactosidase assay for the cells transfectedwith the pSR alpha and pTK constructs after 56 hours. In the case of TKconstructs formation of lacZ- positive blue colonies could be detected,however in the case of SRalpha constructs only single, strongly stained,blue cells could be detected in the culture indicating that plasmids arenot inherited to each of the daughter cells during cell division.

[0092]FIG. 13A shows a number of colonies per 10000 cells transfectedwith equal molar amounts of vectors selected for G418.

[0093]FIG. 13B is an analysis of the episomal DNA in the cellstransfected and selected for G418 with the TK-E1 constructs.

[0094]FIG. 14A is a schematic representation of the designed E2 pointmutations and chimeric E2 proteins.

[0095]FIG. 14B shows an immunoblots of the E2 proteins.

[0096]FIG. 15A shows the transient replication properties of the mutatedE2 proteins in CHO cells.

[0097]FIG. 15B shows the structure of the reporter plasmids used. Thenumbers indicate nucleotide positions in the BPV URR sequence. pUCAluwas used for transient replication studies. pPCAT and pSV3BS9CAT are theCAT reporter plasmids used in transcriptional activation assays.

[0098]FIG. 16 represents a comparison of transactivation and DNA bindingabilities of E2 protein mutants.

[0099]FIG. 17 shows results of a transient transcription assay for theE2 protein mutants. A. Mutations with nearly wild-type properties intransient transcription assay. B. Mutations, which transcriptionalactivity has decreased to 50% of that of the wild-type protein. C.Inactive mutations for transcription. D. Transcriptional properties ofchimeric proteins p53:E2 and VPI6:E2.

[0100]FIG. 18 shows results of staining for beta-galactosidase activityin brain striatum sections, where panels A-F represent plasmid DNAdissolved in cerebrospinal fluid (A-D) or PBS (E, F).

[0101]FIG. 19 is a bar graph showing results of β-galactosidase reportergene expression in mice after injection of a vector of the inventioncontaining a β-galactosidase gene.

[0102]FIG. 20 provides the nucleotide and amino acid sequence of theBPV1 E2 sequence (SEQ ID NO: 1). The positions which were mutated toproduce E2 point mutants are designated as * and are referred to in thetext by amino acid residue. The E2 protein sequence begins at the METindicated as 1.

[0103]FIG. 21 provides the nucleotide sequence of the BPV upstreamregulatory region (SEQ ID NO: 2). The MME sequence is located betweenpositions 7475(Cla 1 site) and the Hpa 1 site (7947).

DESCRIPTION

[0104] The invention is based on the recognition that DNA replication inpapillomavirus from the minimal origin (MO) per se is not sufficient forlong-term persistence, but that in addition another viral sequence isrequired. This sequence, termed herein a minichromosomal maintenanceelement (MME) comprises a binding site for proteins which are essentialfor papillomavirus replication. Although the MME sequence may includebinding sites for replication proteins that are of viral or humanorigin, for example, in BPV, the sequence appears to be dependent onviral proteins E1 and E2, and is specifically bound by the viral E2protein, when the sequence is linked to the minimal origin sequence.

[0105] The invention thus is based on the discovery of a cis-actingelement, referred to herein as a minichromosomal maintenance element(MME), which confers long-term stability to a transiently replicatingeukaryotic episomal plasmid. An MME is distinct from a minimal origin ofreplication (MO), and is required in addition to an MO for long-termplasmid persistence in a host cell.

[0106] Recombinant Vectors of the Invention

[0107] The invention encompasses a genetic construct comprising a singleplasmid containing the MO and MME sequences (MO/MME vectors) and acloning site for insertion of a gene of interest. The invention alsoencompasses a genetic construct comprising a single vector containinggenes encoding E1 and E2 plus MO and MME sequences (E1/E2/M0/MMEvectors) and a cloning site for insertion of a gene of interest. In itsmost useful aspect, the invention features the above described plasmidswherein a gene of interest also is contained in the plasmid.

[0108] Optionally, vectors of the invention may include multiple cloningsite cassettes and selectable markers conferring drug resistance tomammalian and bacterial cells, and reporter genes such as lacZ (FIG. 7).These vectors can be used as stable expression vectors in a wide rangeof both dividing and non-dividing (post-mitotic) cell types.

[0109] An important property of a plasmid containing these determinantsis that replication of this episomal plasmid is not subject toregulation by the cellular controls which regulate host genomereplication; that is, replication occurs independently and is underE1/E2 control in the S-phase of the cell cycle. (FIG. 8). The initiationof E1/E2-dependent replication of MO/MME- containing plasmids is randomin the cell cycle but over-replication does not occur; stable copynumber is maintained in both G1 and G2. (FIG. 9).

[0110] Vectors of the invention are safe to use in human cells andimpart no known oncogenic properties to recipient cells. Allpapilloma-encoded oncogenic sequences have been deleted. (FIGS. 2 and3).

[0111] Definitions

[0112] As used herein, “papillomavirus” refers to a member of thepapilloma family of viruses, including but not limited to bovinepapillomavirus (BPV) and human papillomavirus (HPV).

[0113] “Minimal origin of replication” (MO) refers to a minimalcis-sequence within a papillomavirus that is necessary for initiation ofDNA synthesis. The MO of BPV-1 is located at the 3′ end of the upstreamregulatory region within a 60μ fork, corresponding to a 52 base pair DNAfragment (7928-7947/1-25) including an AT-rich region, a consensussequence to which all papilloma viral E2 proteins bind, and an E1protein binding site spanning nucleotide 1. The MO of HPV is located inthe URR fragment (for example, in HPV11 at nt 7072-793 3/1-99) (Chianget al. PNAS 1992).

[0114] In a transient replication assay, the efficiencies of replicationof plasmids bearing a Minimal Origin (MO) and a full size Origin ofReplication are equal. Only two viral proteins, E1 and E2, are requiredfor stable replication of the full size Origin. An observation which ledin part to the discovery which forms the basis of the invention is thatthe minimal origin of replication (MO) is absolutely essential, but itis not sufficient to stably maintain the plasmids in an episomal state;additional elements in the Upstream Regulatory Region are not onlyrequired, but are sufficient, for stable persistence of the plasmids(FIG. 2).

[0115] “E1” refers to the protein encoded by nt 849-2663 of BPV subtype1; or to nt 832-2779 of HPV of subtype 11, or to equivalent E1 proteinsof other papillomaviruses, or to functional fragments or mutants of apapillomavirus E1 protein, i.e., fragments or mutants of E1 whichpossess the replicating properties of E1.

[0116] “E2” refers to the protein encoded by nt 2594-3837 of BPV subtype1; or to nt 2723-3823 of HPV subtype 11, or to equivalent E2 proteins ofother papillomaviruses, or to functional fragments or mutants of apapillomavirus E2 protein, i.e., fragments or mutants of E2 whichpossess the replicating properties of E2. Numerous E2 mutants aredescribed herein which are defective in the E2 transcriptionalactivating activity and competent in the replicating ability.

[0117] “Minichromosomal maintenance element” (MME) refers to a region ofthe papilloma viral genome to which viral or human proteins essentialfor papilloma viral replication bind, which region is essential forstable episomal maintenance of the papilloma viral MO in a host cell.Preferably, the MME is a sequence containing multiple binding sites forthe transcriptional activator E2, although the MME also may be asequence containing host cell factor binding sites. An MME is mostlikely to be located in the upstream regulatory region of the viralgenome. The MME in BPV is herein defined as the region of BPV locatedwithin the upstream regulatory region which includes a minimum of aboutsix sequential E2 binding sites, and which gives optimum stablepersistence with about ten sequential E2 binding sites, and which mayinclude as many as about 20-30 or as many as about 50 sequential E2binding sites. The sequential binding sites which constitute the MMEneed not be identical in sequence, but must be able to bind E2. Inaddition, between each sequential binding site in the MME, there may bespacer nucleotides, for example, 6 nucleotides, sufficient to insert arestriction enzyme site. The spacer nucleotides may be absent from theMME or may extend to a length of 10, 20 or even 50 nucleotides, so longas the binding of E2 to each separate binding site is not disrupted bythe presence of the spacer.

[0118] The Minichromosome Maintenance Element (MME) of BPV comprisesmultiple, binding sites for the E2 protein (FIG. 3). 10-20 tandemrepeats of E2 binding sites impart greater stability and higher plasmidcopy number (approx. 30 copies per cell, FIG. 4) in cells expressing theBPV (or HPV) E1 and E2 proteins. (FIG. 5). It is believed thatMME/M0-bearing plasmids function in a wide range of eukaryotic cellsincluding rodent, monkey and human cells, and in almost any tissue type.

[0119] The MME confers (E1+E2)-dependent stable replication upon plasmidtransfection into a host cell line expressing both E1 and E2. It isobserved that neither E1 nor E2 alone is sufficient to permitMME-mediated plasmid stability (FIG. 6). E1 and E2 can be provided tothe plasmid either in cis or in trans (i.e. from integrated E1 and E2expression vectors or from the same episomal plasmid).

[0120] “E2 binding site” refers to the minimum sequence ofpapillomavirus double-stranded DNA to which the E2 protein binds. Thisbinding site is in most papillomaviruses located in the upstreamregulatory region in BPV and HPV. In BPV, the E2 binding site is apalindromic 12 nucleotide sequence (ACCN6GGT, where N is any nucleotide)which is repeated approximately 10 times within the URR. The affinitiesof the 10 E2 binding sites for E2 varies among the binding sites in theBPV URR, with site 9 (ACCGN4CGGT, where N is any nucleotide) being thestrongest E2 binding site.

[0121] A “host cell” which is stably transformed according to theinvention may be any prokaryotic or eukaryotic cell, an is preferably amammalian cell, and most preferably a human cell. The cell may bederived from any tissue, for example, muscle, nerve tissue, etc.

[0122] When the E1 and/or E2 genes are located in cis with respect tothe MO and MME, this refers to a genetic context in which one or bothgenes are located on the same episomal element or the same vector as theMO or MME In contrast, when the E1 and/or E2 genes are located in transwith respect to the MO and MME, this refers to a genetic context inwhich the E1 and/or E2 genes are not located on the same episomalelement as the papilloma MO or MME, such as a context in which the E1and/or E2 genes are integrated into the host cell chromosome or the E1and E2 genes are carried on a separate (non-contiguous) genetic element(vector or episome).

[0123] “Stable maintenance” or “long-term persistence” refers to twocharacteristics of vectors of the invention. First, it refers to theability of a vector according to the invention to persist or bemaintained in undividing cells or in progeny cells of dividing cells inthe absence of continuous selection over the long-term. As used herein,“long-term” refers to a period of time that is longer than at leastabout 5 weeks, for example longer than 8 weeks (where a given celldoubling time is about 16 hours). Of course, for a longer or shortercell doubling time, the definition of “long-term” changes accordingly.Second, it refers to the ability of a vector according to the inventionto persist without an appreciable loss of copy number from one celldivision to the next. In determining whether a given vector is capableof long-term persistence, the recombinant vector may be introduced intothe host cell under conditions in which the vector is selected for, whenthe selection conditions are thereafter removed, the copy number of therecombinant vector nevertheless remains constant and reliablethereafter; for example, in non-dividing cells, a vector of theinvention persists for a period that is longer than at least 5 weeks,for example, 6 weeks, 8 weeks, 12 weeks or longer, for a host cell whichdoubles in about 16 hours. A vector of the invention is capable ofpersisting, in dividing cells, over about at least 50 generations, 60generations, 80 cell generations, or 120 cell generations or longerunder non-selective conditions without an appreciable loss of copynumber. We have found that loss of copy number over 80 generations ofcell doublings is less than about 10% (over 80 generations). Therefore,loss of copy number from one cell generation to the next is less thanabout 0.125% or about 0.1%. Therefore, the vectors of the invention arenot subject to an appreciable loss of copy number from one cellgeneration to the next. In contrast, “short-term persistence” of aplasmid in a host cell refers to the inability of a plasmid to persistin a host cell long-term, as defined herein, without an appreciable lossof plasmid copy number, as defined herein.

[0124] A “gene of interest” refers to a gene encoding a gene product ofinterest such as a protein of interest or an RNA of interest. A “proteinof interest” refers to any therapeutic, prophylactic or marker proteinuseful according to the invention. In addition to any therapeuticprotein selected for a treatment using a vector of the invention, atherapeutic protein may include a cell or viral surface antigen to whichan immune response may be elicited when used in a vaccine.

[0125] “Heterologous” or “erogenous” gene refers to a coding sequencethat is introduced into a host cell on a vector of the invention. Thecoding sequence may be identical to a sequence contained within the hostcell, or it is most likely not identical to a host cell sequence.

[0126] “Heterologous promoter” refers to a promoter that is not thenatural (or homologous) promoter that initiates transcription from thegene with which it is associated, whether that gene be the gene encodinga protein of interest or the genes encoding the E1 and E2 proteins. A“strong” promoter, with respect to E1 and E2 expression, refers to apromoter which supports overexpression of the E1 and/or E2 gene to anextent that the vector is maintained in sufficiently high copy number soas to make the host cells unhealthy. An example of a strong promoter isthe SR-alpha promoter; other strong promoters will provide about thesame level of expression of a reporter gene in an assay for quantitatinggene expression and thus promoter strength, e.g., a betagalactosidaseassay.

[0127] As explained hereinabove, a vector useful in the invention willcontain papillomavirus MO and MME sequences, as defined herein, and acloning site for insertion of a gene of interest, and will require thepresence of the papillomavirus E1 and E2 proteins for long-termpersistence in a host cell. The E1 and E2 proteins are effective intrans in the cell and therefore the genes encoding these proteins may bepresent in trans with respect to the vector, or they may be present incis, i.e., within the vector DNA. In addition to the above-describedsequences, a vector useful in the invention may contain a heterologousgene encoding a protein of interest and also may contain sequences forregulation of that gene.

[0128] Described below are experiments in which the BPV MME is localizedand characterized. This experimental strategy also is useful forlocalizing and characterizing the HPV MME. Following the description ofthe BPV MME characterization is a description of the components ofvectors of the invention; i.e., heterologous genes of interest invectors of the invention, heterologous regulatory sequences, e.g.,promoters, which may be used to direct E1 and E2 gene expression and todirect heterologous gene expression, E2 mutants useful according to theinvention, host cells useful in the invention, and delivery vehiclesuseful for delivering vectors of the invention to host cells. TheExamples provided hereinbelow describe specific embodiments of theinvention and are meant to illustrate and not to limit the applicabilityof the invention.

[0129] Localization of BPV MME Sequence

[0130] We have constructed a cell line CHO4.15 expressing constitutivelythe viral proteins E1 and E2, that are required for initiation of viralDNA replication. It has previously been demonstrated that the minimalorigin of replication and the viral E1 and E2 proteins are sufficientfor plasmid replication. It is demonstrated herein that the E1 and E2viral proteins are not only necessary, but are sufficient when coupledwith the MME for long-term episomal persistence.

[0131] Using the cell line CHO4.15 it is shown herein that the BPV-1minimal origin of replication (MO) is absolutely necessary, but is notsufficient for stable extrachromosomal replication of viral plasmids. Bydeletion and insertion analysis, an additional element (MinichromosomeMaintenance Element—MME) in the Upstream Regulatory Region of BPV-1 hasbeen identified which assures stable replication of the MO containingplasmids. This element is composed of multiple binding sites for thetranscriptional activator E2. MME appears to function in the absence ofreplication but requires E1 and E2 proteins for activity. In contrast toEBV or EBV oriP-containing plasmids, for example, stably maintainedBPV-1 plasmids are not subject to once-per-cell cycle replication asdetermined by density labeling experiments. These results indicate thatpapillomavirus episomal replicators replicate independently of thechromosomal DNA of their hosts.

[0132] Construction of Cell Lines Expressing E1 and E2 Proteins

[0133] The E1 and E2 proteins of BPV-1 are necessary for initiation ofDNA replication from the viral origin of replication. Expression ofthese two proteins from heterologous expression vectors allowsreplication of a minimal origin in transient replication assays ( Ustavand Stenlund, 1991; Ustav et al., 1991). However, due to the lack ofpersistence of the transfected expression vectors, replication can notbe monitored for more than few days after transfection.

[0134] To determine whether additional trans-acting factors orcis-acting elements are required for long term persistence of the viralDNA, continuous expression of these two factors has to be assured. Wetherefore constructed several cell lines constitutively expressing theE1 and E2 proteins. Expression of these proteins was directed fromintegrated constructs for E1 protein from CMV promoter (cell lineCHO212) and for E2 protein from HAP 70 promoter (cell line CHO49). Inthe cell line CHO4.15 which expresses both E1 and E2, the E1 protein wasexpressed from SRα-promoter and the E2 protein from HSP 70 promoter.Selection of the respective cell lines and amplification of theexpression units of interest was achieved by utilizing the glutaminesynthetase minigene from the PSVLGS.1 plasmid according to the protocoldescribed earlier (Bebbington and Hentschel, 1987). Expression of E1 andE2 was identified by immunoprecipitation using specific rabbitpolyclonal sera (data not shown) and by in vivo replication assays. Thethree cell lines and the parental CHO cells were transfected with theBPV-1 origin containing plasmnid pUC/Alu in combination with E1 and E2expression vectors. The cell line CHO4.15 which expresses both E1 andE2, supports replication of the origin plasmids in the absence oferogenous E1 and E2. The E2 expressing cell line, CHO49, supportsreplication in the presence of an E1 expression vector, but fails to doso without erogenous E1. The E1 expressing cell line, CHO212, supportsreplication only in the presence of an E2 expression vector. In theparental CHO cell line, co-expression of both E1 and E2 is required forreplication. No replication of pUC/Alu can be detected in the absence ofE1 and E2.

[0135] Described below are experiments which demonstrate the trans andcis-acting elements which are not only necessary but are sufficient forlong-term episomal persistence. The E1 and E2 proteins are bothnecessary and sufficient for stable replication of the BPV-I origincontaining plasmids. In addition, the BPV-I URR contains sequences whichare not only required, but are sufficient for long-term replication ofthe papilloma-derived episome.

[0136] This experimental strategy and procedure also may be used todetermine the exact location in HPV of the MME, simply by substitutingHPV for BPV, and testing regions of the HPV genome. Such testing wouldbegin with the URR of HPV, which region includes binding sites for E2and for cellular factors involved in replication.

[0137] The experimental protocol used for defining the BPV MME was asfollows.

[0138] To determine what viral trans-acting factors and cis-sequenceswere required for long term replication of BPV-1, we used the CHO4.15cell line which constitutively expresses both the viral E1 and E2proteins. Different fragments of BPV-1 were cloned into the vectorpNeo5′ (Lusky and Botchan, 1984 ). This plasmid provides amino glycoside3′-phosphotransferase as a marker for selection of the cells in thepresence of geneticine (G418). We used a 2.5 kb Bg1II fragment from theBPV-1 genome (nucleotides 6946-1515) as a starting fragment. Thisfragment contains the URR including the E2 dependent transcriptionalenhancer, the minimal origin of replication and part of the early ORF's(construct 12, FIG. 2A). This plasmid, in parallel with a minimal originfragment in the same backbone (construct 13, FIG. 2A), were transfectedinto the CHO4.15 cell line by electroporation and processed according tothe scheme in FIG. 1A.

[0139] In FIG. 1A, both transient and long-term plasmid persistence wasdetermined. A portion of the transfected cells were used after platingfor analysis of transient replication. The remaining portion of thecells were selected in the presence of G418 for three weeks, colonieswere then pooled or picked and grown under nonselective conditions fortwo additional weeks, to give a total of five weeks, at which time lowmolecular weight DNA was harvested and analyzed for the presence ofreplicated plasmid. The ability of the origin-containing plasmids toreplicate extra chromosomally in transient and long term replicationassays was examined by Southern analysis of the episomal DNA (seeMaterials and Methods for details). The two plasmids containing the 2.5kb Bg1II fragment and the minimal origin respectively, replicated tocomparable levels in the transient replication assay (FIG. 1B). Afterselection in the long term replication assay, however, the result wasvery different. While the plasmid containing the 2.5 kb Bg1II fragmentcould be readily detected in episomal form, the minimal origincontaining plasmid could not be recovered (compare lanes 12 and 13, FIG.2B). These results showed that sequences present in the larger plasmid,but absent in the plasmid containing only the minimal origin wererequired for long term replication, and that an activity in addition toreplication was required for stable persistence of a plasmid in theCHO4.15 cells. These results also indicated that it was possible tomaintain BPV origin containing plasmids for an extended period of time(e.g., five weeks) without the regulatory circuit that the viral genomecan provide.

[0140] To determine what sequences within the Bg1II fragment wereresponsible for maintenance in the long term assay, we generatedmutations within that fragment and assayed these plasmids formaintenance. Initially, we generated mutations in the sequencessuggested previously to have effects on replication. The BPV-1 Bg1IIfragment contains coding sequences for three potential proteins, E6, E7and the N-terminal part of the E1 protein. Mutations which interruptedE1 ORF, E6 ORF and E6/7 ORF (Lusky and Botchan, 1985; Schiller el al.,1984; Berg et al., 1986)—construct 9 (pNeo Sma⁻), construct 10(pNeo775), construct 11 (pNeo576), respectively and in addition adeletion removing all coding sequences—construct 7 (pNeoBg140), wereintroduced into the pNeo5′ (FIG. 2A). None of these mutations had adetectable effect on maintenance (compare lane 12 with lanes 7, 9, 10and 11 FIG. 2B) indicating that the coding sequences contained withinthe Bg1II fragment were dispensable. Consequently the E1 and E2 are theonly viral gene products required for maintenance.

[0141] It has been suggested previously that BPV-1 URR contains twopartially overlapping cis-regulatory control elements for stablereplication, termed Plasmid Maintenance Sequence (PMS-1) (Lusky andBotchan, 1984) and Negative Control of Replication (NCOR-1) (Roberts andWeintraub 1986). In order to demonstrate that these two sequences werenot necessary or sufficient for long-term plasmid persistence, eachsequence was deleted by removing the sequence between the HindIII andMluI sites (nt. 6959-7351, construct 8—pNeoΔNCOR, FIG. 2A). Thisdeletion had no deleterious effect on long term replication of theplasmid (lane 8, FIG. 2B), demonstrating that these putative elementswere not required. Finally, as a negative control, an XhoI linkerinsertion mutant overlapping with HpaI site was generated (construct 6,FIG. 2A). This mutation generated an origin defective for replication inthe transient replication assay, and it is also defective for long termreplication (lane 6, FIG. 2B). We concluded from these results thatcis-elements required for stable replication of BPV-1 are located withinthe URR and are unrelated to the previously proposed elements PMS-1 andNCOR-1. We have named this cis-element in the BPV-1 URR MinichromosomeMaintenance Element (MME).

[0142] Minichromosomal Maintenance Elements of the Invention

[0143] A minichromosome maintenance element is localized and defined asfollows.

[0144] The Minichromosome Maintenance Element is Composed of RedundantSequences.

[0145] To define the sequences required for long term replication wegenerated a series of deletion mutants within the URR (FIG. 3A). Thesedeletions were made in the context of the plasmid pNeoBg140 (BPV-1sequence from 6946 to 63), and the deletion mutants were tested in thelong term replication assay. The first series of constructs (1-6, FIG.3A) had a fixed 5′-end (nucleotide 7187) and progressive deletions weremade at the 3′-end of the URR (nucleotides 7892, 7834, 7771, 7475, 7389,respectively). The first four of these deletions (constructs 1-4, FIG.3A) were defective for stable replication (FIG. 3B), but the plasmidswith less extensive deletions (plasmids 5 and 6, FIG. 3A) weremaintained at the wild type level (lane 5 and 6, FIG. 3B). Another setof deletions (constructs 7-11, FIG. 3A) had a fixed 3′-end (nucleotide7890) and progressive deletions from the 5′-end (7476, 7611, 7673, 7771,7834, respectively). Two mutants of this series were unable to replicatestably (deletions 7 and 8, FIG. 3A and 3B), but plasmids with lessextensive deletions replicated efficiently (lanes 9-11, FIG. 3A and 3B).The results from the unidirectional deletions showed that a sequence inthe vicinity of nucleotide 7600 was required for long term replication(compare constructs 4 and 5, and 8 and 9). To map this sequence moreprecisely we generated a third set of deletions. These deletions wereconstructed as scanning deletions (constructs, lanes 11-21, FIG. 3A,3B). Surprisingly, none of the introduced mutations resulted in loss ofMME activity demonstrating that no single unique sequence within the URRwas required, but that maybe some redundant sequence element wasresponsible.

[0146] The MME is Composed of Binding Sites for the E2 TranscriptionActivator.

[0147] To address directly if the sequences in the vicinity ofnucleotides 7600 were responsible for the MME activity we generated afragment between nucleotides 7590 and 7673, and inserted this fragmentinto the deletion mutant D221/234 (construct 1 in FIG. 3A) to determineif replication in the long term replication assay could be restored(this fragment corresponds to the sequence between the deletionend-points D 134 and D11). This fragment inserted in three and sixcopies restored MME activity in the long term replication assay (FIG. 5,compare lanes 1 with 2,3). A known constituent of this fragment arethree high affinity E2 binding sites. A possibility that occurred to uswas that the MME activity was contributed by E2 binding sites. Thiswould be consistent with the apparent redundancy, since the URR contains10 binding sites for E2. To determine if E2 binding sites were involvedin MME activity we oligomerized a high affinity E2 binding site 9 of theBPV-1 URR (5′-ACCGTTGCCGGT-3′) with six nucleotide spacing (Li et al.,1989) and inserted these oligomers (10 copies) into D2211234 deletionmutant. This insertion restored the MME activity (lane 4, FIG. 5). Torule out involvement of other BPV sequences we added 10 oligomerized E2binding sites to the minimal origin of replication. Those constructsreplicated with similar efficiency as plasmids with wild type BPV-1sequences in the stable assay (lane 5, FIG. 5). However, plasmids withless than six additional oligomerized E2 binding sites failed toreplicate in the long term replication assay (data not shown). Theseresults strongly suggest that binding sites for the E2 protein can beresponsible for providing MME activity to the BPV-1 origin.

[0148] The MME Enhances the Frequency of Formation of G418-resistantColonies Without Replication.

[0149] It has previously been observed that for EBV, multimerized EBNA-2binding sites (Family of Repeats—FR) in an EBNA-1 dependent fashion arerequired for stable replication of oriP containing plasmids (Krysan etal., 1989; Kirchmaier and Sugden, 1995; Middleton and Sugden, 1994).This activity can be measured by increased transformation frequency ofthe plasmids carrying FR, and is thought to be caused by enhancednuclear retention of plasmids containing FR. To determine if a similaractivity could be determined for MME we measured the transformationfrequency of four different plasmids. First, pNeo5′ carries theselectable neomycin resistance marker, but lacks BPV sequences andconsequently is defective for replication in both the short term andlong term replication assays. Second, the minimal origin plasmid, inaddition carries the BPV minimal origin and is replication competent inthe short term replication assay but not in the long term assay. Thethird plasmid pNeoHpaI→XhoI carries the whole Bg140 fragment, and isthus nominally capable of maintenance, but because of the linkerinsertion in the E1 binding site the plasmid is defective forreplication. The fourth plasmid pNeoBg140 is replication competent inboth the short and long term replication assays. We used CHO4.15 cellsto measure transformation frequency of these different plasmids (FIG.6). The vector with the selectable marker only and the plasmidcontaining the minimal origin transformed CHO4.15 cells with similarfrequency, 14 and 16 colonies per 10⁵ cells, respectively. In theparallel experiment, pNeoHpal→XhoI which is replication defective with amutant E1 binding site, but carrying the sequences required for MMEactivity, transformed CHO4.15 cells 4 to 5 times more efficiently thanvector alone or a plasmid with minimal origin of replication (68.6colonies per 10⁵ cells) (FIG. 6A). The plasmids with the complete origintransformed cells with approximately 100 times higher efficiency thanthe vector containing only the minimal origin of replication(approximately 1600 colonies per 10⁵ cells). These results indicatedthat MME activity could be measured in a stable transformation assayeven in the absence of replication. When the same experiments were alsoperformed in CHO212 (E1 cell line) and CHO49 (E2 cell line), allplasmids transformed with approximately the same efficiency in these twocell lines (FIGS. 6B and 6C). We conclude that enhanced transformationactivity requires both E1 and E2 proteins.

[0150] We measured the possible effect of an MME consisting ofoligomerized E2 binding sites on plasmid retention in the short termassay, as it has been done with the EBV oriP containing plasmids.However, attempts to reproduce a direct effect on nuclear retention intransient assay failed to show a significant effect (data not shown).

[0151] BPV Origin Plasmids Replicate Approximately at 15 Copies perHaploid Genome.

[0152] One of the factors that is expected to influence the stablepersistence of a plasmid is the copynumber. We therefore performedexperiments to estimate the average number of episomal copies perhaploid genome in different established cell lines. After digestion withthe single-cut restriction endonuclease (Hind III) total DNA from threeindependent cell lines-pNeoBg 1 40, pNeo41/Cla and pNeoSca/234 wasloaded in equal amounts onto the gel and was analyzed by Southernblotting using radioactively labeled BPV-1 origin and neo probe. Allthree cell lines contained approximately the same number of episomalplasmids—15 copies per haploid genome (FIG. 4A). Even though no specificeffort was made to determine the number of integrated copies, digestionwith a non-cutter enzymes did not change the appearance of the threeforms and oligomers of the plasmid (FIG. 4B). Consistent with previousreports, the majority of the plasmids were present in the oligomericform. We conclude from these results that the plasmids are mostlyepisomal in the CHO4.15 cell line under the conditions used.

[0153] Mode of Replication

[0154] One explanation for the apparent high stability of BPV-1 plasmidsin the cells could be that the plasmids are subject to the cellular onceper cell cycle replication control. To determine if this was the case weperformed density labeling experiments using the cell line CHO4.15containing the replicating plasmid pNeoBg140 . The experiments wereperformed by continuous labeling of the cells with BrdU for 3.5, 9.5, 15or 24 hours. Low molecular weight DNA and total chromosomal DNA wereextracted after each time point and analyzed by CsCl gradientcentrifugation, followed by slot blotting, and hybridization withplasmnid probe or genomic DNA probe to identify the peaks in thegradient. The density gradient profiles are shown in FIG. 8. The data issummarized in the table as fractions of Bg140 DNA and CHO chromosomalDNA that had incorporated no BrdU (light-light—LL), BrdU incorporatedinto one strand (heavy-light—HL), or into both strands (heavy-heavy—UH)in the CHO4.15 cells stably transformed by this plasmid (FIG. 8). Afterlabeling with BrdU for 3.5 hours (panels a) and e)) the episomal BPV-1origin containing plasmids were divided between three forms of DNA: 5%heavy-heavy, 19% heavy-light and 76% light-light, while chromosomal DNAis distributed between two forms 27% heavy-light and 73% light-light.After labeling for 9.5 hours, the plasmid has accumulated considerableamount (21%) of heavy-heavy DNA, while chromosomal DNA shows nodetectable signal in the heavy-heavy area. After labeling for 15 hours,distribution of the episomal DNA is 34% heavy-heavy, 38% heavy-light,28% light-light. At the same time chromosomal DNA showed still verylittle, if any, heavy-heavy DNA. After labeling for 24 hours, episomalDNA was preferentially in the heavy-heavy fraction of the DNA (66%), 24%heavy-light and 10% light-light, while chromosomal DNA showedconsiderable amount of heavy-heavy DNA (24%), but with most of the DNAstill in the once replicated DNA fraction. These results are consistentwith a doubling time of approximately 16 hours for the pNeoBg140containing cell lines. The considerable percentage of the unreplicatedchromosomal DNA after 24 hours is likely to be due to growth arrest of afraction of the cells by the conditions used for BrdU labeling. Itappears clear from these results that the stably maintained pNeoBg140plasmid does not replicate once per cell cycle and stable persistence ofthe BPV-1 plasmids is not a function of once per cellcycle replicationcontrol.

[0155] Mechanism of Action

[0156] Three mechanisms of action can be envisioned for the MME. First,the MME could affect the efficiency of initiation of replication.Although no difference in replication initiation can be detected duringthe time-course of the transient replication assay (Ustav et al., 1991),it is conceivable that a gradual accumulation of methylated residues atthe origin of replication or some other form of modification (includingnucleosome occlusion) could prevent initiation of replication andresults in gradual loss of replication activity. It is possible that MMEcan affect these processes and prevent inactivation of the origin.Alternatively, the minimal origin containing plasmids are competent forover replication during the S-phase of the cell cycle, which could betoxic to cells. MME could prevent this process, in analogy to thefunction of iterons for certain bacterial plasmids (for reviewNordström, 1991). Third, MME could influence the partitioning processthereby affecting the loss rate of plasmids during cell division.

[0157] It is interesting to note that the ability of the minimal origincontaining plasmids to replicate appears to have no detectable effect ontransformation frequency. One might have expected that an increase inthe quantity of plasmid DNA in the cells as a result of replicationwould lead to a higher frequency of integration. However, this appearsnot to be the case, possibly because these minichromosomes are poorsubstrates for the required recombination events or are lost with veryhigh frequency at cell division. The very large increase intransformation frequency of the plasmids with both MO and MME comparedto integrating marker presumably reflects the fact that the twofunctions together can bypass the requirement for integration possiblyby providing an efficient segregation/partitioning function in additionto replication.

[0158] Materials and Methods

[0159] The following methods are routinely used in the invention. Thesemethods are stated in terms of the detailed experiments performedherein. However, each method may be generalized by one of skill in theart for use in carrying out the invention in its broadest sense, asdescribed below and claimed.

[0160] Plasmid construction. (i) Expression vectors. The E1 and E2protein expression vectors pHSE2 (Szymanski and Stenlund, 1991), pCGE2and pCGEag (Ustav and Stenlund, 1991) have been described earlier. TheE1 expression vector pE1—1×5 contains the BPV-1 E1 ORF with XhoI linkers(within nucleotides 619 to 2757) and carries a point mutation at thesplice donor site at nucleotide 1235. This fragment was cloned into theXhoI site downstream of the SRα-promoter in the plasmid pBJ5GS (kindgift from Dr. L. Berg).

[0161] (ii) Origin plasmids. All origin fragments of the BPV-1 genomewere cloned in sense orientation into the BamHI site of the pNeo5′(Luskyand Botchan, 1984). pNeoBg1II contains a Bg1II fragment from BPV-1 (nt.6946 to 1515) cloned in the sense orientation relative to thetranscription of the neo gene. pNeoXhoI→Hpal contains the same Bg1IIfragment with an XhoI linker insertion into the HpaI site ( Ustav etal., 1991). pNeo 576, pNeo775 and pNeoSma⁻ plasmids contain the sameBg1II fragment with the mutations 576, 775 and Sma- which affect E6/7ORF, E6 ORF and the 5′-part of the E1 ORF, respectively (Lusky andBotchan, 1985; Schiller et al., 1984; Berg et al., 1986). pNeoΔNCOR hasa deletion between HindIlI (nt. 6958) and Mlul (nt.7351). pNeoBg140contained a BPV-1 fragment from nucleotide 6946 to 63, which wasamplified by PCR using respective primers and cloned in senseorientation into the BamHI site. pNeoMO contained minimal originsequence (nucleotides 7914 to 27) cloned into the BamHI site. Linkerdeletion mutants of the BPV-1 genome (Szymanski and Stenlund, 1991) wereused as templates for PCR. Primers—5′-AAAAGCTTTCTTTGGACTTAGA-3′ (BPV-1nucleotides 6959-6979) and 5′-ATAGCCAGCTAACTATAGATCT-3′ (BPV-1nucleotides 45 to 63 flanked by Bg1II site) were used to amplify originfragments. PCR products were cloned into the HindIII and BamfHI site ofthe pNeoBg140. Deletion mutants lacked following sequence:D221/234-7187/7892; D36/234 7187/7834; D121/234-7187/7771;D134/234-7187/7673; DCla/234-7187/7892; DSca/234 7187/7389;D221/Cla-7476/7892; D221/11-7611/7892; D221/134-7673/7892; D221/1217771/7892; D221/36-7834/7892; D36/121-7771/7834; D121/134-7673/7771;D134/43-76227673; D43/11-7611/7622; D11/229-7597/7611;D229/Cla-7476/7597; DCla/41-7355/7476; D41/136-7344/7356;D136/Nar-7273/7344; DNar/64-7214/7273 and junctions contained 8-merBamfHI Tinkers; D221/234+134/1 1×3 contains an insertion of the fragment7590-7673 in three copies and D221/234+134/11×6 in six copies.D221/234+10BS9 has an insertion of the E2 protein binding site 9 in tencopies. DHindIII/221+10BS9 is a deletion between nucleotides 6959/7892which carries 10 copies of oligomerized E2 binding site 9. All deletionand insertion mutants were verified by sequencing.

[0162] Construction of cell lines. CHO-K1 (Chinese Hamster Ovary—ATCCCCL 61) was used as the parental cell line to express BPV-1 replicationproteins.

[0163] (i) E2 cell line CHO49. The E2 expression vector pHSE2 waslinearized with XhoI and the plasmid pBJ5GS carrying glutaminesynthetase minigene expression unit (Bebbington and Hentschel 1987).pBJ5GS (kind gift of Dr. L. Berg) was linearized with SalI endonuclease.The plasmids were mixed at a 1:1 ratio and ligated into the concatemersat high DNA concentration (300 μg/ml) overnight at 16° C. using T4 DNAligase.

[0164] Ten micrograms of the ligated DNA was mixed with 5O μg carrierDNA and was electroporated into the 7×10⁶ CHO-K1 cells using Ham's F12medium supplemented with 10% fetal bovine serum at 22 OV, using a BioRadelectroporation apparatus at the capacitance setting 960 μF. Selectionfor glutamine synthetase was done at 25 μM concentration of theL-methionine sulfoximine (Sigma) in glutamine free Glasgow MinimalMedium supplemented with dialyzed fetal bovine serum, non-essentialamino acids, glutamic acid, aspartic acid, sodium pyruvate, nucleosides,penicillin and streptomycin, essentially as has been described byBebbington and Hentschel (Bebbington and Hentschel, 1987). Colonies werepicked ten days after the selection, expanded and used for the secondround of selection at 250 μM of L-methionine sulfoximine. This step wasincluded to amplify the sequences coupled to the selection marker. Celllines were expanded and tested for expression of E2 protein byimmunoprecipitation with polyclonal rabbit antibodies against E2 protein(Ustav and Stenlund, 1991) after labeling with ³⁵S L-methionine usingTranslabel (ICN) and by functional transient replication assay asdescribed below.

[0165] (ii) E1 cell line CHO212. E1 protein expression vector pCGEag(Ustav and Stenlund 1991) was linearized by the XhoI and pBJ5 GS waslinearized, mixed at a 1:1 ratio and ligated into the concatemers andthe cell line expressing E1 protein was generated essentially same waylike E2 expressing cell line.

[0166] (iii) E1 and E2 expressing cell line CHO4.15. E1 proteinexpression vector pE1-1×5 containing glutamine synthetase minigene andE1 coding sequence was linearized with Sall and pHSE2 was linearizedwith the XhoI restriction endonuclease. Linear plasmids were ligatedinto the concatemers at a ratio 1:1, CHO-K1 cell were transfected byelectroporation and selected as described above.

[0167] Transient replication assays were done as described earlier(Ustav and Steniund, 1991) using the respective cell lines. For testingthe cell lines in functional assay for expression of the BPV-1replication proteins we used 50 ng of pUC/Alu (Ustav et al., 1991) asorigin containing plasmid and 250 ng of the E1 expression vector pE1-1×5or pCGE2 to complement E2 cell line CHO49 or E1 cell line CHO212,respectively. All pNeo5′ based origin plasmids were tested for theirability to replicate in the CHO4.15 cell line by transfecting 100 ng ofthe plasmid DNA together with 50 ptg of denatured carrier salmon spermDNA into the CHO4.15 cells at 24 OV by electroporation. ExtrachromosomalDNA was extracted from the cells at 48 and 72 hours post-transfection byalkaline lysis as described earlier (Ustav and Stenlund, 199 1). DNA waspurified, digested with DpnI and linearizing enzyme and were analyzed bySouthern analysis. Specific probes for hybridization were made by randompriming.

[0168] Stable replication. CHO4.15 cells were electroporated with 100 ngof origin containing plasmid DNA in the presence of 5O μg of carrierDNA. Ninety six hours after transfection CHO4.15 cells were trypsinizedand subjected for the antibiotic G418 selection at the concentration 450μ/ml. Colonies were pooled or single colonies were picked after twoweeks, expanded and episomal or total DNA were analyzed by Southernblotting.

[0169] Copy-number measurement. Total DNA from established cell lines,containing replicating BPV-1 origin plasmids, was extracted and digestedeither with a plasmid single-cutter (HindIll) or a plasmid non-cutter(ApaI), followed by electrophoresis in 0.7% agarose/TAE gels. Thecopynumber was measured by Southern blotting, using probe containingsequences from the BPV ori and neo gene. Results were quantitated bycomparing band intensity to a two-fold dilution series of plasmid DNAusing phosphoimager.

[0170] BrdU labeling and analysis of the replication mode. Cells werelabeled with 35 μg/ml BrdU in MEM medium, containing 2′-deoxycytidine(20 μg/ml) using procedures described earlier (Yates and Guan, 1991).Episomal and chromosomal DNA was extracted at time points indicated inResults section. CsCl solution was added to DNA preparation to 1.74 g/mland centrifuged for 48 hours at 37000 rpm. 24 fractions were collectedfrom each gradient, subjected to denaturation and neutralization, andslot-blotted onto nylon filters. Filters were hybridized with labeledBPV-1 or CHO genomic DNA probes. Radioactivity was counted withPhosphoimager, Fuji.

[0171] Nuclear retention measurement by stable transformation assay. 48hours following transfection the cells were trypsinized, counted andplated at 3 different dilutions. G418 selection was applied, and theselective medium was changed every 3 days. Colonies were counted after10 days.

[0172] Treatment of Diseases According to the Invention

[0173] The invention is useful in vivo and ex vivo human gene therapywhere correction of inherited or acquired genetic defects is desired,and is therefore useful to treat any disease where gene deliveryprovides benefit, whether the gene is delivered to a terminallydifferentiated host cell, such as a hepatocyte, or to anundifferentiated cell such as a stem cell. The invention is useful intreatment of chronic or acute diseases, e.g., T-cell diseases,inflammation, fibroses of the liver, and arthritis. The invention alsois useful in vaccination protocols where resistance or immunity toinfectious pathogens, such as HIV, Hepatitis C Virus, Hepatitis B virus,is desired, or the elimination or induced quiescence of aberrant cells,such as cancer cells, is considered beneficial.

[0174] Recombinant vectors of the invention are useful in that theypermit persistent expression of a therapeutic gene in both dividing andnon-dividing cells; for example, in differentiated cells, such as thosein gut, brain, and muscle.

[0175] Recombinant vectors of the invention are also useful for highlevel transient expression in cells where desired, such as for cancertherapy or in vivo vaccination.

[0176] Both in vivo and ex vivo gene therapy strategies are possiblewith this vector system, including stable, multicopy gene maintenanceand expression, in haemopoietic and other stem cells, and in thecommitted and differentiated progeny of these cell types.

[0177] Nucleic acid vectors suitable for use in the present inventioninclude circular and linear lengths of DNA.

[0178] For human gene therapy, uses of the recombinant vectors of theinvention are not limited in terms of delivery of the vector to a cell.That is, vectors of the invention may be delivered to a cell vianon-viral or viral delivery systems. Delivery systems of non-viralorigin include those which employ molecular conjugates, cationicliposomes, or synthetic peptides, where vector size constraints do notlimit the nature and number of plasmnid vector components. Deliverysystems of viral origin include viral particle-producing packaging celllines as transfection recipients for the above E1/E2/M0/MME-containingplasmids into which viral packaging signals have been engineered, suchas those of adenovirus, herpes viruses and papovaviruses.

[0179] Recombinant vectors of the invention also are useful intransgenesis, including production of transgenic animals via pronuclearinjection, or embryonic stem cell transfection and embryo chimerageneration.

[0180] Heterologous Genes Useful According to the Invention

[0181] Heterologous genes useful in vectors of the invention may alsoencode antigenic determinants of viruses, so as to be useful asvaccines, such antigenic determinants as are present in coat proteins offlu viruses, malaria, TB, and HIV. For modulation of physiologicactivity in brain cells, genes including the following may be ofinterest: CCKA, CCKB, CREB, TH, NT3, NT4, BDNF, GDNF, and NGF.

[0182] Regulatory Sequences Useful According to the Invention

[0183] As described below, regulatory sequences which may be carried invectors of the invention will of course include heterologous promoterand enhancer sequences which control expression of the heterologous geneof interest, and which may be confer a tight level of regulation uponthe heterologous gene, including inhibition or activation of geneexpression, promoter/enhancer strength, tissue specificity, orrelatively little regulation beyond the initiation of transcription.

[0184] Vectors useful according to the invention may advantageouslyinclude a gene encoding a therapeutic agent, a promoter which directsexpression of the gene, and optionally a cellular-derived generegulatory element which confers tissue specific gene expression. Thepromoter/gene combination may be subject to any one of numerous forms ofgene regulation known in the art, for example, production of the geneproduct may be subject to continuous inhibition by associated factorsand thus may require the presence of an activator; alternatively, thegene product may be continuously expressed, and only inhibited undercertain conditions. Gene expression may be regulated at either thetranscriptional or translational level. Where such regulation istranscriptional, it may be at the level of the promoter or at the levelof RNA elongation or processing. Therefore, vectors usefuil according tothe invention may include a heterologous promoter/gene combination thatis turned-on and turned-off in trans by the presence or absence of aregulatory factor.

[0185] Heterologous Regulatory Sequences for E1 and E2 Gene Expression

[0186] In addition to heterologous sequences to regulate a heterologousgene of interest which is carried on the episomal vector, heterologousregulatory sequences are also used in the invention to controlexpression of the papilloma E1 and E2 genes. Proper control ofexpression of E1 and E2 is critical for determining plasmid copy number,stability and segregation, and therefore the invention encompassesmaintenance of an MO+MME-containing plasmid in eukaryotic cells. Thepresence of heterologous regulatory elements has been found to influencepersistent expression, expression in different cell types, andexpression in vivo.

[0187] Choice of a heterologous promoter to drive E1 gene expression hasbeen found to confer certain advantages to a vector of the invention,depending upon the intended use of the construct. For example, it hasbeen found that a strong promoter such as SRalpha (a hybrid of SV40early region and HTLV-1 LTR) to drive expression of the E1 gene can beused advantageously according to the invention to drive plasmid copynumber very high, that is, to levels which render the host cellunhealthy and prevent normal cell division. This generates unstable,onion skin-like products and leads to cell inviability (FIG. 10).However, a strong promoter is advantageously used where high leveltransient expression of a vector heterologous gene is desired, forexample, in treatment of a cancerous condition where it is desirable toproduce a high level of heterologous gene product (for example, a toxin)and it is also desirable to kill the host cell (for example, tumorcells). This type of vector of the invention takes advantage of theability of the MO/MME vector system to replicate at very high levelsindependently of the cell cycle.

[0188] In contrast, it has also been found that use of a weakerpromoter, such as the thymidine kinase promoter, to drive E1 geneexpression can be used advantageously in the invention to permitsufficient E1 protein expression to regulate stable (MO+MME)-containingplasmid replication at high efficiency. (FIG. 10). Similarly, use of theweaker promoter such as thymidine kinase promoter permits E1 proteinsynthesis to a level compatible with efficient expression of a reportergene product from the constructs in transient assay (FIG. 11). Theresults shown in FIG. 12 demonstrate stable expression of beta-gal inthe cells upon selection. SRalpha-E1 constructs express reporter,however cells are not dividing while Tk-E1 constructs work forexpression in transient as well as in stable cells; cells dividenormally to give colonies which stain blue for beta-gal protein.

[0189] Proper control of post-transcriptional processing of E1 and E2transcripts further influences the replicative and expression propertiesof the plasmid system (efficiency, compatibility with host replicationsystem, stability, toxicity), and is improved by incorporation ofsequences such as those of human beta globin gene (exxonII, intronII,exonIII sequences), and SV40 sequences which incorporate splice, polyAand mRNA stability sequences. Improved expression vectors harboring suchelements in the E1 and E2 expression domains are described herein. FIGS.13A and 13B show G418 selection of different vectors (SRalpha-E1 andTkE1 constructs).

[0190] In addition to the use of regulatory sequences for regulation ofE1 and E2 gene expression, the invention also encompasses the use ofregulatory sequences for control of a heterologous gene encoding aprotein of interest, which gene is carried on an episomal vector of theinvention.

[0191] The adenovirus E1A promoter and enhancer, the human CMV-MIEpromoter and enhancer, retroviral LTR elements, herpesvirus promoters,and poxvirus promoters are representative examples of heterologousregulatory elements useful in the invention.

[0192] Modification of a heterologous promoter useful according to theinvention may be accomplished according to a number of strategies. Forexample, the use of negative regulatory elements which decrease thelevel of transcription is envisaged. Especially preferred, however, isthe modification of the promoter sequences in order to reduce the basallevels of transcription. Promoter sequences may be modified in order toremove regulatory elements which respond to cell-specific regulatoryfactors. Preferably, therefore, elements responsible for activation bycell-specific factors may be mutated or deleted.

[0193] Modified Forms of a Promoter Useful in the Invention

[0194] The invention encompasses the use of modified forms of aheterologous promoter, which exhibit decreased levels of basaltranscription. Such heterologous promoters may be usefuil in theinvention, because it may be desirable to confer upon the host cell alow enough level of transcription of, for example, an anti-viral gene ora gene encoding a toxin so as to prevent deleterious effects on the hostcell by virtue of the presence of the encoded gene product in the cell.

[0195] Modified forms of a given promoter may be made, as is well-knownin the art, using conventional PCR and incorporating random or directedbase substitutions, deletions, or insertions in the native promotersequence.

[0196] Transactivatable viral promoters useful according to theinvention include but are not limited to the following: Herpes Virus(HSV-1), immediate early promoter (Liv et al., 1990, Biotechniques9:168; Rice et al., 1990, Jour. Virol. 64:1704; Howard et al., 1993,Exp. Cell. Res. 207:194); Visna Virus promoters (Carroth et al., 1994,Jour. Virol. 68:6137); Papillomavirus promoters (Storey et al., 1990,Jour. Ge. Virol. 71-965 and Ibaraki et al., 1993, Virus Genes 7:187;Epstein-Barr Virus promoters (TPI and Barn HIC promoters) Cohen et al.,1991, Jour. Virol. 65-5880; Sung et al., 1991 Jour. Virol. 65:2164; andMeitinger et al., 1994, Jour. Virol. 68:7497); CMV promoters (IE2, IE1,and MIE promoters) (Malone et al., 1990, Jour. Virol. 64:1498; Cockettet al., 199 1, Nucl. Ac. Res. 19:319; and Pizzomo et al., 1991, JourVirol. 65:3839); Hepatitis B promoters (Seto et al., 1990, Nature344:72; Raney et al., 1991, Jour. Virol. 65:5774; Lauer et al., 1994,Hepatology 19:23); Spumaretroviral promoters (Rethwillm et al., 1990,Virol. 175:568, Venkatesn et al., 1993, Jour. Virol. 67:3868); HTLV-1promoters (Franklin et al., 1993, Jour. Biol. Chem. 268:21225; Kadisonet al., 1990, Jour. Virol. 64:2141); and Adenoviral promoters (E2)(Obert et al., 1994, Mol. All. Biol. 14:1333).

[0197] Heterologous promoters useful according to the invention,including modified forms of a given promoter, are first tested for basalactivity using a reporter gene, and are also tested to determine if theypossess or retain the ability to be trans-activated. Where a vector ofthe invention includes heterologous promoter/gene combination which ispresent in the cell in an inhibited state and which requirestransactivation for production of the encoded heterologous gene product,such promoter/gene combinations may be most useful provided they possessor retain the ability to be transactivated while exhibiting a low enoughlevel of basal activity such that the gene product is virtuallyundetectable.

[0198] A vector of the invention, whether its heterologous promoter ismodified or unmodified, may be tested for its ability to betrans-activated to the extent that differential killing of transformedcells occurs. Functional tests of diminished basal activity from vectorsof the invention will include the use of prodrug activating systems,such as ganciclovir, in cells containing a vector in which theheterologous promoter/gene combination is in the inhibited versus theactivated state.

[0199] Regulatory Elements conferring Cell-Type Specific HeterologousGene Expression

[0200] Cellular-derived genetic elements also may be included inepisomal vectors of the invention.

[0201] These genetic elements thus are responsive to, i.e., subject tocontrol by, cellular factors. Tissue-specific promoters may confertissue specificity.

[0202] E2 Mutants Defective for Transcriptional Activation

[0203] The invention also encompasses the use of E2 mutants which aredefective in the E2 transcriptional activator function. A gene encodingsuch an E2 mutant is incorporated into the episomal vector system of theinvention.

[0204] Full-length E2 protein has two functional domains which arewell-conserved among the E2 proteins of different papollomaviruses, a200 amino acid transactivation domain at the amino terminus and a 90-100amino acid carboxy terminal domain that is essential for dimerizationand DNA binding. A flexible spacer hinge region separates thetransactivation and DNA binding domains.

[0205] In order to provide an E2 mutant which is defective intranscriptional activation, but which are able to supply the E2 functionthat is necessary and sufficient for episomal persistence, pointmutations have been made in the gene encoding this protein. Thesemutations are useful where they eliminate the transcriptional activatingactivity of the protein without affecting the ability of the resultantprotein to stably maintain (MO+MME)-containing plasmids in transfectedcells.

[0206] E2 mutants were made by selected mutation of amino acids whichare believed to lie in hydrophilic domains at the protein surface, andby then changing basic or acidic amino acids to a small hydrophobicand/or polar residue, e.g., alanine, which has a short side chainconsisting of a single —CH₃ group; serine, which also contains a singlecarbon atom side chain (—CH₂OH); valine, leucine, threonine andisoleucine, which are hydrophobic amino acids having side chains rangingfrom 2-4 carbon-containing groups. The mutation is introduced into theE2 gene using oligonucleotide-directed mutagenesis in M13 phage. It iswithin the scope of the invention to locate additional point mutationswhich abolish the transcription activating activity of E2 withoutdeleteriously affecting its replication function to maintain episomalvectors according to the invention.

[0207] Manufacture of Vectors of the Invention

[0208] The invention also features vectors which include sequencesconferring replication and selection in lower eukaryotic or prokaryotichost cells in order to manufacture a usefuil quantity of vector DNA,e.g., 100 ug-mg quantities.

[0209] Additional sequences which may be present in a vector of theinvention to enable manufacture of vector DNA include other origins ofreplication, e.g., a bacterial or yeast origin of replication, whichpermits preparation of vector DNA in prokaryotic or eukaryotic cells, orbaculovirus sequences which allow for vector replication in insectcells.

[0210] This aspect of the invention is applicable to most strains ofbacteria, for example, gram positive and negative bacterial strains, andyeast. Gram negative bacteria useful according to the invention includebut are not limited to E. coli and Salmonella, e.g., S. typhimurium.Gram positive species useful according to the invention include but arenot limited to Bacillus and Lactococcus.

[0211] Prokaryotic and Eukaryotic Origins of Replication

[0212] The invention can be utilized advantageously with a plasmidorigin of replication that permits replication of at least 10,preferably at least 20-100, and most preferably at least 200-500 copiesof the plasmid per host cell. Those origins of replication that permitreplication of moderate (i.e., 2050) to high plasmid (i.e., 200-500)copy numbers are especially useful. Of course, if desired, a plasmidhaving a copy number, as high as 1000-2000 copies per cell, also may beused.

[0213] Plasmids with low copy numbers (i.e., 10 copies or less) are mostadvantageously used according to the invention after mutation to bringabout increased copy number (J. Scott, 1984, Microbial Reviews 48:1-23).Of the frequently used origins of replication, pBR322 (20 copies/cell)is useful according to the invention, as is pUC (at 200 copies/cell).Other plasmids whose origins of replication may be useful according tothe invention are those which require the presence of plasmid encodedproteins for replication, for example, the pT181, FII, and FI origins ofreplication.

[0214] Examples of origins of replication which are useful according tothe invention in E. coli and S. typhimurium include but are not limitedto pMB1 (25 or more copies per cell, Bolivar et al., 1977, Gene2:95-113), ColE1 (15 or more copies per cell, Kahn et al., 1979, MethodsEnzymol. 68:268280), p15A (about 15 copies per cell, Chang et al., 1978,J. Bacteriol. 134:1141-1156); pSC101 (about 6 copies per cell, Stoker etal., 1982, Gene 18:335-341); R6K (less than 15 copies per cell, Kahn etal., 1979, supra); R1 (temperature dependent origin of replication,Uhlin et al., 1983, Gene 22:255-265); lambda dv (Jackson et al., 1972,Proc. Nat. Aca. Sci. 69:2904-2909). An example of an origin ofreplication that is useful in Staphylococcus is pT181 (about 20 copiesper cell, J. Scott, 1984, Microbial Reviews 48:1-23. Of theabove-described origins of replication, pM1, p15A and ColE1 arepreferred because these origins do not require plasmid-encoded proteinsfor replication.

[0215] Genes Encoding Selectable Marker Useful in Vectors of theInvention

[0216] Genes encoding selectable markers useful in vectors of theinvention include antibiotic resistance genes, for example encodingresistance to antibiotics such as ampicillin, kanamycin or tetracycline,are the most common dominant selectable markers used in molecularbiology cloning and fermentation procedures for the production ofrecombinant proteins or plasmid DNA.

[0217] Preparation of vector DNA can also be used in conjunction withantibiotic resistance. Representative antibiotic resistance genes usefulaccording to the invention include, for example, the kanamycin gene,from pUC4K (Pharmacia Biotech) by restricting the plasmid with XhoI andfilling in the 5′ overhang. This plasmid DNA is then restricted withPstI and the fragment containing the kanamycin gene is then gelpurified. Other useful antibiotic resistance genes are well-known in theart, including the genes encoding chlorainphenicol acetyl transferase,ampicillin, and tetracycline.

[0218] A vector of the invention which is intended for manufacture inbacteria will include in addition to the papillomavirus replicationelements described herein, for example, a bacterial origin ofreplication and a gene encoding a selectable marker. The vector DNA willbe transformed into a bacterial host, and the host grown under selectionconditions. Plasmid DNA is then prepared according to conventionalmeans.

[0219] Delivery of Episomal Vector to Host Cell

[0220] Vectors of the invention may be delivered to a host cell via anyone of a number of vector delivery means. The invention is not limitedby the mode of delivery of the vector to the host cell. Transfer of avector of the invention to a host cell can be accomplished via any ofthe following, including but not limited to direct injection of nakedDNA, for example, using a gene gun, transfection using calcium phosphatecoprecipitation, fusion of the target cell with a liposomal vehicle,erythrocyte ghosts or spheroplasts carrying DNA, plasmid and viralvector-mediated transfer, DNA protein complex-mediated gene transfersuch as receptor-mediated gene transfer, and viral infection.

[0221] Receptor-mediated gene transfer is dependent upon the presence ofsuitable ligands on the surfaces of cells which will allow specifictargeting to the desired cell type followed by internalization of thecomplex and expression of the DNA. One form of receptor-mediated genetransfer is wherein a DNA vector is conjugated to antibodies whichtarget with a high degree of specificity cell-surface antigens (Wong andHuang, 1987, Proc. Nat. Aca. Sci. 84:7851; Roux el al., 1989, Proc. Nat.Aca. Sci. 86::9079; Trubetskoy et al., 1992, Bioconjugate Chem. 3:323;and Hirsch et al., 1993, Transplant Proceedings 25:138). Nucleic acidmay be attached to antibody molecules using polylysine (Wagner el al.,1990, Proc. Nat. Aca. Sci. 87:3410; Wagner et al., 1991, Proc. Nat. Aca.Sci. 89:7934) or via liposomes.

[0222] Increased expression of DNA derived from ligand-DNA complexestaken up by cells via an endosomal route has been achieved through theinclusion of endosomal disruption agents, such as influenza virushemagglutinin fusogenic peptides, either in the targeting complex or inthe medium surrounding the target cell. The fusogenic peptide of the HAmolecule is a modified form of HA which retains two important functionsof HA. It allows for fusion of the targeted DNA/ligand complex to thecell membrane, but without the host cell sialic acid-binding specificityof the natural molecule. Instead, host cell binding specificity isconferred by the ligand/receptor interaction. The modified HA fusogenicpeptide also retains the HA function of endosomal uptake, thus allowingfor uptake of the complex into the host cell via membrane fusion, andthe endosomal escape function of HA, which allows for escape of theenveloped DNA from the endosomal/lysosomal destruction pathway. Thefusogenic peptide may include the HA amino acid sequenceGLFGAIAGFIGAGTGGMIAGGGC.

[0223] 1. Viral Vectors

[0224] Recombinant viral vectors as well as other DNA transfer schemescan be used in practice of the present invention. A recombinant viralvector of the invention will include DNA of at least a portion of aviral genome which portion is capable of infecting the target cells. By“infection” is generally meant the process by which a virus transfersgenetic material to its host or target cell. Preferably, the virus usedin the construction of a vector of the invention is also renderedreplication-defective to remove the effects of viral replication on thetarget cells. In such cases, the replication-defective viral genome canbe packaged by a helper virus in accordance with conventionaltechniques. Generally, any virus meeting the above criteria ofinfectiousness and capabilities of functional gene transfer can beemployed in the practice of the invention.

[0225] Suitable viruses for practice of the invention include but arenot limited to, for example, papovavirus and herpesvirus, well known tothose skilled in the art; suitable vector packaging cell lines forstably transducing mammalian cell lines are known in the art.

[0226] It will be appreciated that when viral vector schemes areemployed for gene transfer according to the invention, the use of anattenuated or a virulent virus also may be desirable.

[0227] 2. Delivery of Gene via DNA-Protein Complexes

[0228] Targeted gene delivery is also achieved according to theinvention using a DNA-protein complex. Such DNA-protein complexesinclude DNA complexes with a ligand that interacts with a target cellsurface receptor. Cell surface receptors are thus utilized as naturallyexisting entry mechanisms for the specific delivery of genes to selectedmammalian cells. It is known that most, if not all, mammalian cellspossess cell surface binding sites or receptors that recognize, bind andinternalize specific biological molecules, i.e., ligands. Thesemolecules, once recognized and bound by the receptors, can beinternalized within the target cells within membrane-limited vesiclesvia receptor-mediated endocytosis. Examples of such ligands include butare not limited to proteins having functional groups that are exposedsufficiently to be recognized by the cell receptors. The particularproteins used will vary with the target cell.

[0229] Typically, glycoproteins having exposed terminal carbohydrategroups are used although other ligands such as antibodies or polypeptidehormones, also may be employed. Using this technique the phototoxicprotein psoralen has been conjugated to insulin and internalized by theinsulin receptor endocytotic pathway (Gasparro, Bio-chem. Biophys. Res.Comm. 141(2), pp. 502509, Dec. 15, 1986); the hepatocyte specificreceptor for galactose terminal asialoglycoproteins has been utilizedfor the hepatocyte-specific transmembrane delivery ofasialoorosomucoid-poly-L-lysine non-covalently complexed to a DNAplasmid (Wu, G. Y., J. Biol. Chem., 262(10), pp. 44294432, 1987); thecell receptor for epidermal growth factor has been utilized to deliverpolynucleotides covalently linked to EGF to the cell interior (Myers,European Patent Application 86810614.7, published Jun. 6, 1988); theintestinally situated cellular receptor for the organometallic vitaminB₁₂-intrinsic factor complex has been used to mediate delivery to thecirculatory system of a vertebrate host a drug, hormone, bioactivepeptide or immunogen complexed with vitamin B₁₂ and delivered to theintestine through oral administration (Russel-Jones et al., Europeanpatent Application 863 07849.9, published Apr. 29, 1987); themannose-6-phosphate receptor has been used to deliver low densitylipoproteins to cells (Murray, G. J. and Neville, D. M., Jr., J. Bio.Chem. Vol. 225 (24), pp. 1194-11948, 1980); the cholera toxin bindingsubunit receptor has been used to deliver insulin to cells lackinginsulin receptors (Roth and Maddox, J. Cell. Phys. Vol. 115, p. 151,1983); and the human chorionic gonadotropin receptor has been employedto deliver a ricin a-chain coupled to HCG to cells with the appropriateHCG receptor in order to kill the cells (Oeltmann and Heath, J. Biol.Chem, Vol 254, p. 1028 (1979)). Ligands selected from biotin, biotinanalogs and biotin receptor-binding ligands, and/or folic acid, folateanalogs and folate receptor-binding ligands to initiate receptormediated transmembrane transport of the ligand complex, as described inU.S. Pat. No. 5,108,921.

[0230] Generally, a ligand is chemically conjugated by covalent, ionicor hydrogen bonding to the nucleic acid. A ligand for a cell surfacereceptor may be conjugated to a polycation such as polylysine withethylidene diamino carbodiimide as described in U.S. Pat. No. 5,166,320.DNA may be attached to an appropriate ligand in such a way that thecombination thereof or complex remains soluble, is recognized by thereceptor and is internalized by the cell. The DNA is carried along withthe ligand into the cell, and is then expressed in the cell. The proteinconjugate is complexed to DNA of a transfection vector by mixing equalmass quantities of protein conjugate and DNA in 0.25 molar sodiumchloride. The DNA/protein complex is taken up by cells and the gene isexpressed.

[0231] Delivery of the foreign DNA into the target cell may also beachieved via the DNA construct's association with an endosomaldisruption agent, such as the influenza hemagglutinin fusogenic peptide,as described above.

[0232] 3. Liposomal Gene Transfer

[0233] Liposomes have been used for non-viral delivery of manysubstances, including nucleic acids, viral particles, and drugs. Anumber of reviews have described studies of liposome productionmethodology and properties, their use as carriers for therapeutic agentsand their interaction with a variety of cell types. See, for example,“Liposomes as Drug Carriers,” Wiley and Sons, NY (1988), and “Liposomesfrom Biophysics to Therapeutics,” Marcel Dekker, NY (1987). Severalmethods have been used for liposomal delivery of DNA into cells,including poly-L-lysine conjugated lipids (Zhou et al., Biochim.Biophys. Acta. 1065:8-14, 1991), pH sensitive immunoliposomes(Gregoriadis, G., Liposome Technology, Vol I, II, III, CRC, 1993), andcationic liposomes (Felgner et al., Proc. Natl. Acad. Sci., USA,84:7413-7417, 1987). Positively charged liposomes have been used fortransfer of heterologous genes into eukaryotic cells (Felgner et al.,1987, Proc. Nat. Aca. Sci. 84:7413; Rose et al., 1991, BioTechniques10:520). Cationic liposomes spontaneously complex with plasmid DNA orRNA in solution and facilitate fusion of the complex with cells inculture, resulting in delivery of nucleic acid to the cell. Philip etal. 1994, Mol. and Cell. Biol. 14:2411, report the use of cationicliposomes to facilitate adeno-associated virus (AAV) plasmidtransfection of primary T lymphocytes and cultured tumor cells.

[0234] Delivery of an agent using liposomes allows for non-invasivetreatment of diseases. Targeting of an organ or tissue type may be mademore efficient using immunoliposomes, i.e., liposomes which areconjugated to an antibody specific for an organ-specific ortissue-specific antigen. Thus, one approach to targeted DNA delivery isthe use of loaded liposomes that have been made target-specific byincorporation of specific antibodies on the liposome surface.

[0235] Host Cells Useful in the Invention

[0236] The cells targeted for in vivo or ex vivo gene transfer inaccordance with the invention include any cells to which the delivery ofthe therapeutic gene is desired. Eukaryotic cells are preferred, andparticularly mammalian cells. For example, brain cells, cells of thecentral nervous system, nerve cells, gut cells, skin cells, kidneycells, endothelium, lung cells, liver cells, cells of the immune systemsuch as T-cells, B-cells, and macrophages.

[0237] Dosage, Mode of Administration and Pharmaceutical Formulation

[0238] Vector DNA may be formulated as naked DNA for parenteraladministration or as a transfection mixture. In the latter case, thetransfection mixture may be assembled just prior to use. In the case ofa pharmaceutical composition, the vector DNA includes the papillomavirusMO and MME sequences, optionally, the E1 and E2 genes, and a gene whoseexpression is intended to have some beneficial therapeutic effect on thecells of the recipient. For optimal efficiency of delivery of naked DNAto a target tissue, it is preferred that the vector DNA be delivered at10-100 ug/10,000 cells, optimally about 50 ug/10,000 cells.

[0239] The DNA may be exchanged into isotonic phosphate free buffer andsterile filtered, and then aliquotted into suitable vials. The vials maybe stored at 4° C., 20° C. or 80° C. or alternatively the mixture may befreeze dried from a buffer containing an appropriate carrier and bulkingagent. In these cases, the dosage form is reconstituted with a sterilesolution before administration.

[0240] For delivery of vector DNA in vivo or ex vivo, the vectorcontaining a gene of physiological importance, such as replacement of adefective gene or an additional potentially beneficial gene function, isexpected to confer long term genetic modification of the cells and beeffective in the treatment of disease.

[0241] For example, a patient that is subject to a viral or geneticdisease, or who is being vaccinated against a virus or pathogen, may betreated in accordance with the invention via in vivo or ex vivo methods.For example in vivo treatments, a vector of the invention can beadministered to the patient, preferably in a biologically compatiblesolution or a pharmaceutically acceptable delivery vehicle, byingestion, injection, inhalation or any number of other methods. Thedosages administered will vary from gene to gene, disease to disease,and from patient to patient; a “therapeutically effective dose” will bedetermined by the level of enhancement of function of the transferredgenetic material balanced against any risk or deleterious side effects.Monitoring levels of gene introduction, gene expression and/or thepresence or levels of the encoded therapeutic protein will assist inselecting and adjusting the dosages administered. Generally, acomposition including the vector will be administered in a single dosein the range of 10 ng-1 mglkg body weight, preferably in the range of100 ng-100 ug/kg body weight, such that at least one copy of thetherapeutic gene is delivered to each target cell. Vector DNA may beadministered as a single dose, or in multiple doses, as needed. Thetherapeutic gene will, of course, be associated with appropriateregulatory sequences for expression of the gene in the target cell.

[0242] Ex vivo treatment is also contemplated within the presentinvention. Cell populations can be removed from the patient or otherwiseprovided, transduced with a therapeutic gene in accordance with theinvention, then reintroduced into the patient. In general, ex vivo celldosages will be determined according to the desired therapeutic effectbalanced against any deleterious side-effects. Such dosages will usuallybe in the range of 10⁵-10⁸ cells per patient, daily weekly, orintermittently; preferably 10⁶-10⁷ cells per patient.

EXAMPLES

[0243] The following examples describe detailed embodiments of theinvention in which a given gene of interest is introduced and stablymaintained in a specific host cell, or a specific disease is exemplifiedfor treatment according to the invention.

Example 1

[0244] Recombinant Host Cells of the Invention

[0245] Recombinant vectors of the invention may be used in the in vitroproduction of a protein of interest, for example, cell factories may beprepared by transforming a host cell with a recombinant vector of theinvention. The vector will contain the papillomavirus MO, MME and a geneencoding the protein of interest, and may optionally include the genesencoding the HPV or BPV E1 and E2 genes. Alternatively, a cell line maybe prepared that carries in its chromosome the E1 and E2 genes, suchthat the encoded proteins are in trans to the papilloma episomalsequences. A cell line carrying a recombinant vector of the invention,whether it carries the E1 and E2 genes in cis or in trans to the vectorpermits stable, high level expression of proteins of therapeutic valuein cultured mammalian cells.

[0246] Where it is desirable according to the invention to determine thecopy number (per haploid or diploid host cell genome) of a recombinantvector of the invention. Vector copy number may be determined asdescribed herein for copy number determination of the BPVorigin-containing plasmids.

[0247] Described below is an example in which a host cell line isengineered to contain chromosomal copies of the E1 and E2 genes. Thiscell line is advantageous for production of a desired protein or RNA inthat it can be transfected with a vector of the invention encoding thedesired protein or RNA, and cultured to produce that molecule.

[0248] We constructed several cell lines constitutively expressing theE1 and E2 proteins. Expression of these proteins was directed fromintegrated constructs for E1 protein from CMV promoter (cell lineCHO212) and for E2 protein from HAP70 promoter (cell line CHO49). In thecell line CHO4.15 which expresses both E1 and E2, the E1 protein wasexpressed from SRαpromoter and the E2 protein from HSP70 promoter.Selection of the respective cell lines and amplification of theexpression units of interest was achieved by utilizing the glutaminesynthetase minigene from the pSVLGS.1 plasmid according to the protocoldescribed earlier (Bebbington and Hentschel, 1987). Expression of E1 andE2 was identified by immunoprecipitation using specific rabbitpolyclonal sera (data not shown) and by in vivo replication assays. Thethree cell lines and the parental CHO cells were transfected with theBPV-1 origin containing plasmnid pUC/Alu in combination with E1 and E2expression vectors. The cell line CHO4.15 which expresses both E1 andE2, supports replication of the origin plasmids in the absence oferogenous E1 and E2. The E2 expressing cell line, CHO49, supportsreplication in the presence of an E1 expression vector, but fails to doso without erogenous E1. The E1 expressing cell line, CHO212, supportsreplication only in the presence of an E2 expression vector. In theparental CHO cell line, co-expression of both E1 and E2 is required forreplication. No replication of pUC/Alu can be detected in the absence ofE1 and E2.

[0249] The cell line CHO4.15 is transformed with a vector of theinvention, for example, the pBN/TKE1-0 vector shown in detail in FIG.7B. Alternatively, where transient high level expression of the gene ofinterest is desired, the vector pBNE1, shown in FIG. 7A may be used toproduce relatively higher quantities of the protein of interest. We haveobserved MO/MME vector persistence in CHO4.15 cells for at least 80 cellgenerations with a copy number loss less than 10% over 80 generations.Each of these vectors contains the beta-galactosidase reporter gene.However, as is described below, the beta-gal gene can be removed andsubstituted with a gene of interest. The transformed cells are grownunder conditions, as described in Materials and Methods, to produce theencoded molecule of interest.

Example 2

[0250] E2 Transcriptional Activator Mutants

[0251] The invention also encompasses the use of E2 point mutants whichare defective in the E2 transcriptional activator function and competentin the replication function. Such E2 mutants are incorporated into theepisomal vector system of the invention. Point mutations have been madein the gene encoding this protein which eliminate the transcriptionalactivating activity of the protein without affecting the ability of theresultant protein to stably maintain (MO+MME)-containing plasmids intransfected cells.

[0252] The mutants were made by selected mutation of amino acids whichare believed to lie in hydrophilic domains (i.e., hydrophilic domains ofbetween about 4-15 amino acids) and/or alpha helical domains at theprotein surface. Such mutants are made, for example, by amino acidsubstitutions in the alpha helix region 2 (amino acid residues 23-49)and helix 3 (amino acids residues 62-82), and by then changing basic oracidic amino acids to a small hydrophobic residue. The mutation isintroduced into the E2 gene using oligonucleotide-directed mutagenesisin M 13 phage. It is within the scope of the invention to locateadditional point mutations which abolish the transcription activatingactivity of E2 without deleteriously affecting its replication function.

[0253]FIG. 14A depicts the location of the mutations and schematicallyillustrates the predicted secondary structure (cylinder—helix, shadedbox—sheet) for the N-terminal and the X-ray structure for the C-terminalportions of the E2 protein. The locations of mutations are indicated byarrows; numbers refer to the position of the mutated residue in the E2protein.

[0254]FIG. 14B demonstrates that mutant E2 proteins are stably expressedin cells. Immunoblots of the E2 proteins are shown. Cell lysates wereprepared from COS-7 cells containing a wild-type (WT) or mutant E2expression plasmid. 30 μl of cell lysate was loaded on each lane. Thenegative control lysate was prepared from cells electroporated withcarrier DNA. The substitutions of amino acids are shown on thelettering, and single amino acid code is used; the numbers refer to thepositions of the substituted residues in the E2 protein.

[0255]FIG. 15A shows the replication activation properties of the mutantE2 proteins using transient replication assays of the mutated E2proteins in CHO cells. Cells were electroporated with 100 ng of reporterplasmid pUC/Alu, 500 ng of pCGEag and 250 ng of pCGE2, which expresseswild-type E2 or its derivatives. Cells were harvested either 36 or 48hours after electroporation, low mol.wt. DNA was digested with Dpnl andlinearizing enzyme Hind III and analyzed by the Southern blotting.

[0256]FIG. 15B shows the reporter constructs for determiningtranscription activation properties of the E2 mutants, and originconstructs for determining activation of replication. The numbersindicate nucleotide positions in the BPV URR sequence. pUCAlu was usedfor transient replication studies. pPCAT and pSV3BS9CAT are the CATreporter plasmids used in transcriptional activation assays.

[0257]FIG. 16 shows the transcription activation activity of the mutantE2 proteins and represents comparison of transactivation and DNA bindingabilities of E2 protein mutants. The radioactive signals of gel-shiftassays were quantitated with the use of a Phosphorlmager (data notshown). After scanning, the E2 specific signal of wtE2 protein was setas 1. For transcription assay CHO cells were electroporated with 250 ngof respective reporter and 250 ng of pCGE2 or its derivatives.Normalized CAT activities were determined 40 hours after transfection.In all cases the values shown represent the results of three independenttransfection experiments.

[0258]FIG. 17 demonstrates of the self-squelching feature of some of theE2 mutants at the higher concentrations. Transient transcription assayfor the E2 protein mutants were performed as described in Materials andMethods. CHO cells were transfected with increasing amounts of theexpression vector for E2 mutants or chimeric E2 proteins, as indicated,and with 250 ng of reporter plasmid pSV3BS9CAT. Normalized CATactivities were determined 40 hours after transfection. Each pointrepresents the result obtained in three independent transfection assays.FIG. A. Mutations with nearly wild-type properties in transienttranscription assay. B. Mutations, which transcriptional activity hasdecreased to 50% of that of the wild-type protein. C. Inactive mutationsfor transcription. D. Transcriptional properties of chimeric proteinsp53:E2 and VP16:E2.

[0259] The mutants R37A (i.e., Arg-37-->Ala), E74A (Glutamic Acid-74-->Ala), D122A, D143A/R172C are particularly useful according to theinvention because they are defective or crippled for transcriptionalactivation of promoters are essentially wild type for activation ofreplication in the transient replication assay. Table 1 shows codonswhich were mutated, giving rise to the described amino acid changes.TABLE 1 codon wt mutated start codon codon change name 2716 AGA GCAArg-37 Ala R37A 2827 GAA GCA Glu-74 Ala E74A 3121 CGC TGC Arg-172 CysR172C

[0260] E2 mutants such as those described herein can be tested for theirability to replicate MO+MME containing plasmids using the MO/MMEreplication assays described herein. The use of such E2 mutants isadvantageous in that it eliminates, or at least minimizes, thepossibility of aberrant activation of cellular genes including proto-oncogenes by the E2 protein, and represents an improved safety featureof this system. The safety of the vector is increased by theinactivation of the E2 transcription activation domain. Furthermore, thespecificity of the vector and also its safety is increased because themutant form of E2 would not activate E2 responsive promoters which maybe present on the vector. In addition, the mutant form of E2 describedherein would not transactivate E2 enhancers. It is known that some E2binding sites constitute an E2 enhancer. These binding sites, whenpresent on a vector according to the invention as part of the MME, wouldnot be transactivated by mutant E2, thus further reducing thedeleterious effects of wild-type E2 in this vector system. Use of themutant form of E2 disclosed herein also may improve the specificity oftissue-specific promoters or other promoters which drive expression of atherapeutic (or other) gene on the vector. That is, because promotersmay have some leakiness, transcription from a leaky promoter driving E2gene expression may result in E2 expression and initiation of E2transactivation of an E2-driven promoter and gene. However, theinventive mutant form of E2 is defective in transactivation andtherefore would not initiate E2-responsive transcription even in thepresence of a leaky promoter driving mutant E2 gene expression.Therefore, a tissue-specific promoter which drives expression of a geneof interest on a vector of the invention may be more tightly regulatedas a result of use of an E2 mutant according to the invention.

Example 3

[0261] Stable persistence of Vector of the Invention in Muscle Cells

[0262] The usefulness of the E1/E2/M0/MME episomal vector system for invivo delivery to, and long term expression in, mammalian muscle isdemonstrated as follows. Generation of a humoral immune response togenes expressed in these vectors has been achieved by expression inmuscle by direct intramuscular injection of vector DNA, testifying toits utility as an in vivo vaccine system. Briefly, the E1/E2/M0/MMEplasmid is directly injected into mice intramuscularly into each leg; 50ul saline containing 10 Oug plasmid DNA is injected into the quadracepsmuscle of each leg. Injections were performed in 1, 2, or 3 sets at 3week intervals (Ulmer, 1993, Science 259;1745). FIG. 19 shows theresults of the experiment. 0.1 mg of plasmid DNA was injected into miceintramuscularly. Serum samples were collected at 90 days afterinjection. Antibodies were measured by ELISA using recombinantβ-galactosidase as antigen. The OD at 414 was read using a 1:1000dilution of serum. “Neg. Control” refers to saline alone injection;“pUE83” refers to an RSV LTR promoter-driven β-gal expression cartridgecloned into pUC19; “SRalpha vector” refers to the E1/E2/M0/MME plasmnidcontaining the E1 and E2 genes driven by the SRalpha promoter and thesame β-gal expression cartridge; and “TK vector” refers to theE1/E2/M0/MME plasmid containing the E1 and E2 genes driven by the TKpromoter and the same β-gal expression cartridge. The MME in the SRalphaand TK vectors is the region in the BPV URR corresponding to positions6958-1.

Example 4

[0263] Stable persistence of Vector of the Invention in Brain Cells

[0264] The following example demonstrates persistent, long termexpression of lacZ reporter gene in the central nervous system using anE1/E2/M0/MME vector harboring a lacZ reporter gene (FIG. 18). Briefly,the vector SR-alpha #2 (SR promoter directing E1 and E2 expression inthe E1/E2/M0/MME plasmid described in detail above) was injected into anadult rat brain striatum region (bregma +1.7 dex. +2.5). Total amount ofvector DNA was 50 micrograms in 5 microliter of PBS. Plasmid DNA wasdissolved in PBS (panels E and F) or artificial cerebrospinal fluid(panels A-D). The results showed that P-galactosidase activity wasdetected histochemically by X-GAL regular staining protocol on frozenbrain tissue slices after 5 days (panels A and B), two weeks (panels Cand D) and three months (panels E and F) exposure. Control animals wereinjected with PBS (E) or CSF (panels A and C). Therefore, the plasmnidwas stably maintained in brain tissue for as long as three months afterintroduction.

[0265] The above example demonstrates the utility of vectors of theinvention in gene therapy and in providing animal models of disease andtherapy.

[0266] References

[0267] The following references are referred to hereinabove and theircontents are hereby incorporated by reference.

[0268] Bebbington, C. R. and Hentschel, C. C. G (1987) In Glover, D. M.(ed.), DNA cloning—A practical Approach. IRL Press, Oxford, Vol 111, pp.163-188.

[0269] Berg, L., Lusky, M., Stenlund, A., and Botchan, M. R. (1986)Repression of bovine papillomavirus replication is mediated by a virallyencoded trans-factor. Cell,_(—)46, 753-762.

[0270] Blitz, I. L and Laimins, L. A. (1991) The 68-kilodalton E1protein of bovine papillomavirus is a DNA binding phoshoprotein whichassociates with the E2 transcriptional activator in vitro. J Virol.,65,649-656.

[0271] Bonne-Andrea, C., Santucci, S. and Clertant, P. (1995) Bovinepapillomavirus E1 protein can, by itself, efficiently drive multiplerounds of DNA synthesis in vitro. J Virol., 69, 3201-3205.

[0272] Botchan, M. R., Berg, L., Reynolds, J. and Lusky, M. (1986) Thebovine papillomavirus replicon. In D. Evered and S. Clark (eds.),Papillomaviruses. John Wiley & Sons, New York, pp 5367.

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[0319] Other Embodiments

[0320] Other embodiments will be evident to those of skill in the art.It should be understood that the foregoing detailed description isprovided for clarity only and is merely exemplary. The spirit and scopeof the present invention are not limited to the above examples, but areencompassed by the following claims.

1. A method of obtaining long-term stable production of a gene productof interest in a host cell, comprising providing a host cell containinga vector comprising (A) a minimal origin of replication of a papillomavirus, (B) a minichromosomal maintenance element of a papilloma virus,and (C) a gene encoding said gene product, wherein said vector, whenpresent in a mammalian host cell, persists in said cell for at leastabout 50 cell generations in dividing cells or for at least about 8weeks in non-dividing cells under nonselective conditions without anappreciable loss of copy number.
 2. A method of obtaining long-termstable production of a gene product of interest in a host cell,comprising providing a host cell containing a vector comprisingpapillomavirus sequences consisting essentially of (A) a papillomavirusE1 gene and E2 gene, (B) a minimal origin of replication of a papillomavirus, (C) a minichromosomal maintenance element of a papilloma virus,and (D) a gene encoding said gene product, wherein said vector persistsin said cell for at least about 50 cell generations in dividing cells orfor at least about 8 weeks in non-dividing cells under nonselectiveconditions without an appreciable loss of copy number.
 3. A method ofobtaining long-term stable production of a gene product of interest in ahost cell, comprising providing a host cell containing a pair of vectorscomprising (I) a first vector comprising papillomavirus sequencesconsisting essentially of (A) a papillomavirus E1 and E2 gene, (B) aminimal origin of replication of a papilloma virus, and (C) aminichromosomal maintenance element of a papilloma virus, and (II) asecond vector comprising papillomavirus sequences consisting essentiallyof (A) a gene encoding said gene product, (B) a minimal origin ofreplication of a papilloma virus, and (C) a minichromosomal maintenanceelement of a papilloma virus, wherein said vector persists in said cellfor at least about 50 cell generations in dividing cells or for at leastabout 8 weeks in non-dividing cells under nonselective conditionswithout an appreciable loss of copy number.
 4. Use of a recombinantvector for obtaining long term stable maintenance of erogenous DNA in aeukaryotic host cell wherein the recombinant vector comprises: minimalorigin of replication of a papillomavirus; minichromosomal maintenanceelement of a papillomavirus; and heterologous DNA sequence encoding anexpressible gene.
 5. The method of claims 1-3 or the use of claim 4,said minichromosomal maintenance element of papillomavirus being fromBPV.
 6. The method of claims 1-3 or the use of claim 4, said minimalorigin of replication of papillomavirus being from BPV.
 7. The method ofclaim 1 or the use of claim 4, further comprising a papillomavirus geneencoding E2.
 8. The method of claims 1-3 or the use of claim 4, furthercomprising a papillomavirus gene encoding E1.
 9. The method or use ofclaim 7 or 8 wherein said papillomavirus gene encoding E1 or E2comprises a structural gene encoding E1 or E2 operatively associatedwith regulatory sequences for expression of the structural gene in ahost cell.
 10. The method or use of claim 9, said regulatory sequencescomprising a promoter.
 11. The method or use of claim 10, said promotercomprising a promoter that is non-native to said structural gene. 12.The method or use of claim 11, said promoter being functional in morethan a single tissue type.
 13. The method or use of claim 11, saidpromoter being functionally restricted to a single tissue type.
 14. Themethod or use of claim 11, said promoter comprising one of the thymidinekinase promoter and the SR alpha promoter.
 15. The method or use ofclaim 11, said promoter being a strong promoter.
 16. The method ofclaims 1-3 or the use of claim 4, said vector further comprising abacterial host cell origin of replication.
 17. The method of claims 1-3or the use of claim 4, said vector further comprising a gene encoding aselectable marker.
 18. The method of claims 1-3 or use of claim 4wherein said gene is operatively associated with regulatory sequencesfor expression of the gene in a host cell.
 19. The method or use ofclaim 18, said regulatory sequences comprising a promoter.
 20. Themethod or use of claim 19, said promoter comprising a promoter that isnon-native to said structural gene.
 21. The method or use of claim 20,said promoter being functional in more than a single cell type.
 22. Themethod or use of claim 20, said promoter being functionally restrictedto a single cell type.
 23. The method or use of claim 7 wherein saidgene encodes a mutant form of E2 which is replication competent butdefective in transcriptional activation wherein said mutant form of E2protein differs from the wild-type E2 in a nucleotide point mutationwhich translates into an amino acid substitution.
 24. The method or useof claim 23 wherein said gene encoding said mutant form of E2 isoperatively associated with a cell-type-restricted promoter.
 25. Themethod or use of claim 23 wherein said gene encoding said mutant form ofE2 is in trans to said minimal maintenance element and operativelyassociated with a cell-type-restricted promoter.
 26. The method or useof claim 23, said amino acid substitution being R37A.
 27. The method oruse of claim 23, said amino acid being E74A.
 28. The method or use ofclaim 23, said amino acid being D22A and D143A/R172C.
 29. The method ofclaims 1-3 or the use of claim 4 wherein the minichromosomal maintenanceelement and the minimal origin of replication consists of a DNA sequencethat is different from the natural papillomavirus sequence.
 30. Themethod of claims 1-3 or the use of claim 4 wherein the minchromosomalmaintenance element and the minimal origin of replication are separatedby a distance of less than about 1.0 kb.
 31. The method of claims 1-3 orthe use of claim 4 wherein the minichromosomal maintenance elementconsists essentially of the region of BPV mapping to positions 7590 to7673.
 32. The method of claims 1-3 or the use of claim 4 wherein theminichromosomal maintenance element comprises (BPV E2 binding sites 6, 7and 8) x, wherein x is 3 to
 6. 33. The method of claims 1-3 or the useof claim 4 wherein the ainchromosomal maintenance element comprises atleast 2 of the 3 E2 binding sites 6, 7 and
 8. 34. A vector for use inany one of claims 30-33.
 35. A recombinant vector for stable maintenanceof erogenous DNA in a eukaryotic host cell, the vector comprisingpapilloma virus sequences consisting essentially of (A) a minimal originof replication of a papilloma virus, (B) a minichromosomal maintenanceelement of a papilloma virus consisting essentially of at least two ofthe three E2 binding sites 6, 7, and 8, wherein the region of the vectorcomprising the minimal origin of replication and minichromosomalmaintenance element consists of a DNA sequence different from thenatural papilloma virus sequence, and wherein said vector, when presentin a mammalian host cell which expresses E1 and E2, persists in saidcell for at least about 50 cell generations in dividing cells or for atleast about 8 weeks in non-dividing cells under nonselective conditionswithout an appreciable loss of copy number.
 36. A recombinant vector forstable maintenance of erogenous DNA in a eukaryotic host cell, thevector comprising papilloma virus sequences consisting essentially of(A) a minimal origin of replication of a papilloma virus, and (B) aminichromosomal maintenance element of a papilloma virus consistingessentially of multiple E2 binding sites, wherein the distance betweensaid minimal origin of replication and said minichromosomal maintenanceelement is less than about 1.0 kb, wherein said vector, when present ina mammalian host cell which expresses E1 and E2, persists in said cellfor at least about 50 cell generations in dividing cells or for at leastabout 8 weeks in non-dividing cells under nonselective conditionswithout an appreciable loss of copy number.
 37. A recombinant vector forstable maintenance of erogenous DNA in a eukaryotic host cell, thevector comprising papilloma virus sequences consisting essentially of(A) a minimal origin of replication of a papilloma virus, (B) a minichromosomal maintenance element of a papilloma virus consistingessentially of the region of BPV mapping to about positions 7590-7673wherein said vector, when present in a mammalian host cell whichexpresses E1 and E2, persists in said cell for at least about 50 cellgenerations in dividing cells or for at least about 8 weeks innon-dividing cells under nonselective conditions without an appreciableloss of copy number.
 38. A recombinant vector for stable maintenance oferogenous DNA in a eukaryotic host cell, the vector comprising papillomavirus sequences consisting essentially of (A) a minimal origin ofreplication of a papilloma virus, and (B) a minichromosomal maintenanceelement of a papilloma virus consisting essentially of (BPV E2 bindingsites 6, 7, and 8)x wherein x is 3-6, wherein said vector, when presentin a mammalian host cell which expresses E1 and E2, persists in saidcell for at least about 50 cell generations in dividing cells or for atleast about 8 weeks in non-dividing cells under nonselective conditionswithout an appreciable loss of copy number.
 39. The recombinant vectorof claims 35-38, said minichromosomal maintenance element ofpapillomavirus being from BPV.
 40. The recombinant vector of claims35-38, said minimal origin of replication of papillomavirus being fromBPV.
 41. The recombinant vector of claims 35-38, further comprising anexpressible gene of interest.
 42. The recombinant vector of claims35-38, further comprising a papillomavirus gene encoding E1.
 43. Therecombinant vector of claims 35-38, further comprising an papillomavirusgene encoding E2.
 44. The recombinant vector of claim 42 or 43 whereinsaid papillomavirus gene encoding E1 or E2 comprises a structural geneencoding E1 or E2 operatively associated with regulatory sequences forexpression of the structural gene in a host cell.
 45. The recombinantvector of claim 44, said regulatory sequences comprising a promoter. 46.The recombinant vector of claim 45, said promoter comprising a promoterthat is non-native to said structural gene.
 47. The recombinant vectorof claim 46, said promoter being functional in more than a single celltype.
 48. The recombinant vector of claim 46, said promoter beingfunctionally restricted to a single cell type.
 49. The recombinantvector of claim 46, said promoter comprising one of the thymidine kinasepromoter and the SR alpha promoter.
 50. The recombinant vector of claim46, said promoter being a strong promoter.
 51. The recombinant vector ofclaims 35-38, further comprising a bacterial host cell origin ofreplication.
 52. The recombinant vector of claim 51, further comprisinga gene encoding a selectable marker.
 53. The recombinant vector of claim41 wherein said gene comprises a structural gene operatively associatedwith regulatory sequences for expression of the structural gene in ahost cell.
 54. The recombinant vector of claim 53, said regulatorysequences comprising a promoter.
 55. The recombinant vector of claim 54,said promoter comprising a promoter that is non-native to saidstructural gene.
 56. The recombinant vector of claim 55, said promoterbeing functional in more than a single cell type.
 57. The recombinantvector of claim 55, said promoter being functionally restricted to asingle cell type.
 58. The recombinant vector of claim 43 wherein saidgene encodes a mutant form of E2 which is replication competent butdefective in transcriptional activation, wherein the mutation is a pointmutation.
 59. The recombinant vector of claim 58 wherein said geneencoding said mutant form of E2 is operatively associated with acell-type restricted promoter.
 60. The recombinant vector of claim 58wherein said gene encoding said mutant form of E2 is in trans to saidminimal maintenance element and operatively associated with acell-type-restricted promoter.
 61. A host cell containing the vector ofclaims 35-60.
 62. The host cell of claim 61, said cell being mammalian.63. The host cell of claim 61, said mammalian cell being muscle orbrain.
 64. A method of obtaining stable long-term expression of a geneof interest in a cell, comprising providing the host cell of claim 61.65. A method of obtaining stable expression of a gene of interest in acell, comprising introducing into a host cell the recombinant vector ofclaims 35-38.
 66. A method of treating a disease stemming from a geneticdefect, comprising administering a therapeutically effective amount thevector of claims 35-38 to a patient afflicted with said disease.
 67. Amethod of manufacture of vector DNA, comprising culturing the host cellof claim
 61. 68. A method of producing a protein of interest in a hostcell, comprising culturing the host cell of claim 61 under conditionswhich permit expression of said gene of interest.
 69. A method ofproducing a protein in a transgenic animal, comprising providing atransgenic animal containing the vector of claim 41 which produces theprotein of interest.
 70. A mutant form of a papillomavirus E2 proteinwherein the replication function of said protein is competent and thetranscriptional activation function of said protein is defective,wherein said mutant form of E2 protein differs from the wild-type E2 ina nucleotide point mutation which translates into an amino acidsubstitution.
 71. The mutant form of a papillomavirus E2 protein ofclaim 70, said amino acid substitution being R37A.
 72. The mutant formof a papillomavirus E2 protein of claim 70, said amino acid substitutionbeing E74A.
 73. The mutant form of a papillomavirus E2 protein of claim70, said amino acid substitution being D 122A and D 143A/R172C.
 74. Agene encoding the mutant form of the papillomavirus E2 protein of claim70.
 75. A host cell transformed with the gene of claim
 74. 76. A kit forproviding stable persistence of a vector in a host cell, the kitcomprising the vector of claims 35-38 and packaging materials therefor.77. A kit for providing a stably transformed host cell to a patient, thekit comprising the host cell of claim 61 and packaging materialstherefor.
 78. A kit for providing a mutant E2 protein to a host cell forstable persistence of a vector in the host cell, the kit comprising themutant E2 protein of claim 70, and packaging materials therefor.
 79. Akit for providing a mutant E2 protein to a host cell for stablepersistence of a vector in the host cell, the kit comprising the gene ofclaim 74, and packaging materials therefor.
 80. A mammalian diseasemodel comprising a transgenic animal whose cells contain the vector ofclaim 41.